Patent application title: METHODS FOR QUANTITATING SMALL RNA MOLECULES
Inventors:
Christopher K. Raymond (Seattle, WA, US)
Christopher K. Raymond (Seattle, WA, US)
Assignees:
Merck Sharp & Dohme Corp.
IPC8 Class: AC12Q168FI
USPC Class:
435 611
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid nucleic acid based assay involving a hybridization step with a nucleic acid probe, involving a single nucleotide polymorphism (snp), involving pharmacogenetics, involving genotyping, involving haplotyping, or involving detection of dna methylation gene expression
Publication date: 2012-01-12
Patent application number: 20120009580
Abstract:
In one aspect, the present invention provides methods for amplifying a
microRNA molecule to produce DNA molecules. The methods each include the
steps of: (a) using primer extension to make a DNA molecule that is
complementary to a target microRNA molecule; and (b) using a universal
forward primer and a reverse primer to amplify the DNA molecule to
produce amplified DNA molecules. In some embodiments of the method, at
least one of the forward primer and the reverse primer comprise at least
one locked nucleic acid molecule.Claims:
1. A kit for detecting at least one human microRNA selected from the
group consisting of miR-21, miR-22, miR-33, miR-34a, miR-34b, miR-34c,
miR-122, and miR-122a, the kit comprising at least one or more
oligonucleotide primers selected from the group consisting of SEQ ID
NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID
NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ
ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ
ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.
2. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73 and SEQ ID NO:74 for detecting miR-21.
3. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:449 and SEQ ID NO:450 for detecting miR-22.
4. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:111 and SEQ ID NO:112 for detecting miR-33.
5. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO: 113,and SEQ ID NO:114 for detecting miR-34a.
6. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:116 for detecting miR-34b.
7. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:117 and SEQ ID NO:118 for detecting miR-34c.
8. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:20 for detecting miR-122.
9. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:147 and 148 for detecting miR-122a.
10. An oligonucleotide primer for detecting a human microRNA selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No. 11/779,759, filed Jul. 18, 2007, which is a continuation-in-part of application Ser. No. 10/579,029, filed Nov. 19, 2008, which is the National Stage of International Application No. PCT/US2006/002591, filed Jan. 25, 2006, which claims the benefit of Provisional Application No. 60/647,178, filed Jan. 25, 2005, all of which are incorporated herein by reference in their entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 37790_Sequence_Final.txt. The text file is 250 KB; was created on Sep. 20, 2011; and is being submitted via EFS-Web with the filing of the specification.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of amplifying and quantitating small RNA molecules.
BACKGROUND OF THE INVENTION
[0004] RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression (Bernstein et al. (2001), Nature 409:363-6; Dykxhoorn et al. (2003) Nat. Rev. Mol. Cell. Biol. 4:457-67). The phenomenon of RNAi was first described in Caenorhabditis elegans, where injection of double-stranded RNA (dsRNA) led to efficient sequence-specific gene silencing of the mRNA that was complementary to the dsRNA (Fire et al. (1998) Nature 391:806-11). RNAi has also been described in plants as a phenomenon called post-transcriptional gene silencing (PTGS), which is likely used as a viral defense mechanism (Jorgensen (1990) Trends Biotechnol. 8:340-4; Brigneti et al. (1998) EMBO J. 17:6739-46; Hamilton & Baulcombe (1999) Science 286:950-2).
[0005] An early indication that the molecules that regulate PTGS were short RNAs processed from longer dsRNA was the identification of short 21 to 22 nucleotide dsRNA derived from the longer dsRNA in plants (Hamilton & Baulcombe (1999) Science 286:950-2). This observation was repeated in Drosophila embryo extracts where long dsRNA was found processed into 21-25 nucleotide short RNA by the RNase III type enzyme, Dicer (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). These observations led Elbashir et al. to test if synthetic 21-25 nucleotide synthetic dsRNAs function to specifically inhibit gene expression in Drosophila embryo lysates and mammalian cell culture (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). They demonstrated that small interfering RNAs (siRNAs) had the ability to specifically inhibit gene expression in mammalian cell culture without induction of the interferon response.
[0006] These observations led to the development of techniques for the reduction, or elimination, of expression of specific genes in mammalian cell culture, such as plasmid-based systems that generate hairpin siRNAs (Brummelkamp et al. (2002) Science 296:550-3; Paddison et al. (2002) Genes Dev. 16:948-58; Paddison et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1443-8; Paul et al. 2002) Nat. Biotechnol. 20:404-8). siRNA molecules can also be introduced into cells, in vivo, to inhibit the expression of specific proteins (see, e.g., Soutschek, J., et al., Nature 432 (7014):173-178 (2004)).
[0007] siRNA molecules have promise both as therapeutic agents for inhibiting the expression of specific proteins, and as targets for drugs that affect the activity of siRNA molecules that function to regulate the expression of proteins involved in a disease state. A first step in developing such therapeutic agents is to measure the amounts of specific siRNA molecules in different cell types within an organism, and thereby construct an "atlas" of siRNA expression within the body. Additionally, it will be useful to measure changes in the amount of specific siRNA molecules in specific cell types in response to a defined stimulus, or in a disease state.
[0008] Short RNA molecules are difficult to quantitate. For example, with respect to the use of PCR to amplify and measure the small RNA molecules, most PCR primers are longer than the small RNA molecules, and so it is difficult to design a primer that has significant overlap with a small RNA molecule, and that selectively hybridizes to the small RNA molecule at the temperatures used for primer extension and PCR amplification reactions.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) producing a first DNA molecule that is complementary to a target microRNA molecule using primer extension; and (b) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule. It will be understood that, in the practice of the present invention, typically numerous (e.g., millions) of individual microRNA molecules are amplified in a sample (e.g., a solution of RNA molecules isolated from living cells).
[0010] In another aspect, the present invention provides methods for measuring the amount of a target microRNA in a sample from a living organism. The methods of this aspect of the invention include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method including the steps of: (1) producing a first DNA molecule complementary to the target microRNA molecule in the sample using primer extension; (2) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer; and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.
[0011] In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.
[0012] In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer for amplifying the cDNA molecule and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kit, at least one of the universal forward and reverse primers include at least one locked nucleic acid molecule. The kits of the invention may be used to practice various embodiments of the methods of the invention.
[0013] The present invention is useful, for example, for quantitating specific microRNA molecules within different types of cells in a living organism, or, for example, for measuring changes in the amount of specific microRNAs in living cells in response to a stimulus (e.g., in response to administration of a drug).
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
[0015] FIG. 1 shows a flow chart of a representative method of the present invention;
[0016] FIG. 2 graphically illustrates the standard curves for assays specific for the detection of microRNA targets miR-95 and miR-424 as described in EXAMPLE 3;
[0017] FIG. 3A is a histogram plot showing the expression profile of miR-1 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5;
[0018] FIG. 3B is a histogram plot showing the expression profile of miR-124 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5; and
[0019] FIG. 3C is a histogram plot showing the expression profile of miR-150 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0020] In accordance with the foregoing, in one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the universal forward primer and the reverse primer comprises at least one locked nucleic acid molecule.
[0021] As used herein, the term "locked nucleic acid molecule" (abbreviated as LNA molecule) refers to a nucleic acid molecule that includes a 2'-0,4'-C-methylene-β-D-ribofuranosyl moiety. Exemplary 2'-0,4'-C-methylene-β-D-ribofuranosyl moieties, and exemplary LNAs including such moieties, are described, for example, in Petersen, M., and Wengel, J., Trends in Biotechnology 21(2):74-81 (2003) which publication is incorporated herein by reference in its entirety.
[0022] As used herein, the term "microRNA" refers to an RNA molecule that has a length in the range of from 21 nucleotides to 25 nucleotides. Some microRNA molecules (e.g., siRNA molecules) function in living cells to regulate gene expression.
[0023] Representative Method of the Invention. FIG. 1 shows a flowchart of a representative method of the present invention. In the method represented in FIG. 1, a microRNA is the template for synthesis of a complementary first DNA molecule. The synthesis of the first DNA molecule is primed by an extension primer, and so the first DNA molecule includes the extension primer and newly synthesized DNA (represented by a dotted line in FIG. 1). The synthesis of DNA is catalyzed by reverse transcriptase.
[0024] The extension primer includes a first portion (abbreviated as FP in FIG. 1) and a second portion (abbreviated as SP in FIG. 1). The first portion hybridizes to the microRNA target template, and the second portion includes a nucleic acid sequence that hybridizes with a universal forward primer, as described infra.
[0025] A quantitative polymerase chain reaction is used to make a second DNA molecule that is complementary to the first DNA molecule. The synthesis of the second DNA molecule is primed by the reverse primer that has a sequence that is selected to specifically hybridize to a portion of the target first DNA molecule. Thus, the reverse primer does not hybridize to nucleic acid molecules other than the first DNA molecule. The reverse primer may optionally include at least one LNA molecule located within the portion of the reverse primer that does not overlap with the extension primer. In FIG. 1, the LNA molecules are represented by shaded ovals.
[0026] A universal forward primer hybridizes to the 3' end of the second DNA molecule and primes synthesis of a third DNA molecule. It will be understood that, although a single microRNA molecule, single first DNA molecule, single second DNA molecule, single third DNA molecule and single extension, forward and reverse primers are shown in FIG. 1, typically the practice of the present invention uses reaction mixtures that include numerous copies (e.g., millions of copies) of each of the foregoing nucleic acid molecules.
[0027] The steps of the methods of the present invention are now considered in more detail.
[0028] Preparation of microRNA Molecules Useful as Templates. microRNA molecules useful as templates in the methods of the invention can be isolated from any organism (e.g., eukaryote, such as a mammal) or part thereof, including organs, tissues, and/or individual cells (including cultured cells). Any suitable RNA preparation that includes microRNAs can be used, such as total cellular RNA.
[0029] RNA may be isolated from cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein. Cells of interest include wild-type cells, drug-exposed wild-type cells, modified cells, and drug-exposed modified cells.
[0030] Additional steps may be employed to remove some or all of the DNA. Cell lysis may be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. In one embodiment, RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (see, Chirgwin et al., 1979, Biochemistry 18:5294-5299). Separation of RNA from DNA can also be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
[0031] If desired, RNase inhibitors may be added to the lysis buffer. Likewise, for certain cell types, it may be desirable to add a protein denaturation/digestion step to the protocol.
[0032] The sample of RNA can comprise a multiplicity of different microRNA molecules, each different microRNA molecule having a different nucleotide sequence. In a specific embodiment, the microRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences. In other embodiments, the microRNA molecules of the RNA sample comprise at least 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000, or 100,000 different nucleotide sequences.
[0033] The methods of the invention may be used to detect the presence of any microRNA. For example, the methods of the invention can be used to detect one or more of the microRNA targets described in a database such as "the miRBase sequence database" as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/. A list of exemplary microRNA targets is also described in the following references: Lagos-Quintana et al., Curr. Biol. 12(9):735-9 (2002).
[0034] Synthesis of DNA Molecules Using microRNA Molecules As Templates. In the practice of the methods of the invention, first DNA molecules are synthesized that are complementary to the microRNA target molecules, and that are composed of an extension primer and newly synthesized DNA (wherein the extension primer primes the synthesis of the newly synthesized DNA). Individual first DNA molecules can be complementary to a whole microRNA target molecule, or to a portion thereof; although typically an individual first DNA molecule is complementary to a whole microRNA target molecule. Thus, in the practice of the methods of the invention, a population of first DNA molecules is synthesized that includes individual DNA molecules that are each complementary to all, or to a portion, of a target microRNA molecule.
[0035] The synthesis of the first DNA molecules is catalyzed by reverse transcriptase. Any reverse transcriptase molecule can be used to synthesize the first DNA molecules, such as those derived from Moloney murine leukemia virus (MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemia virus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV-RT). A reverse transcriptase lacking RNaseH activity (e.g., SUPERSCRIPT III® sold by Invitrogen, 1600 Faraday Avenue, P.O. Box 6482, Carlsbad, Calif. 92008) is preferred in order to minimize the amount of double-stranded cDNA synthesized at this stage. The reverse transcriptase molecule should also preferably be thermostable so that the DNA synthesis reaction can be conducted at as high a temperature as possible, while still permitting hybridization of primer to the microRNA target molecules.
[0036] Priming the Synthesis of the First DNA Molecules. The synthesis of the first DNA molecules is primed using an extension primer. Typically, the length of the extension primer is in the range of from 10 nucleotides to 100 nucleotides, such as 20 to 35 nucleotides. The nucleic acid sequence of the extension primer is incorporated into the sequence of each, synthesized, DNA molecule. The extension primer includes a first portion that hybridizes to a portion of the microRNA molecule. Typically the first portion of the extension primer includes the 3'-end of the extension primer. The first portion of the extension primer typically has a length in the range of from 6 nucleotides to 20 nucleotides, such as from 10 nucleotides to 12 nucleotides. In some embodiments, the first portion of the extension primer has a length in the range of from 3 nucleotides to 25 nucleotides.
[0037] The extension primer also includes a second portion that typically has a length of from 18 to 25 nucleotides. For example, the second portion of the extension primer can be 20 nucleotides long. The second portion of the extension primer is located 5' to the first portion of the extension primer. The second portion of the extension primer includes at least a portion of the hybridization site for the universal forward primer. For example, the second portion of the extension primer can include all of the hybridization site for the universal forward primer, or, for example, can include as little as a single nucleotide of the hybridization site for the universal forward primer (the remaining portion of the hybridization site for the forward primer can, for example, be located in the first portion of the extension primer). An exemplary nucleic acid sequence of a second portion of an extension primer is 5' CATGATCAGCTGGGCCAAGA 3' (SEQ ID NO:1).
[0038] Amplification of the DNA Molecules. In the practice of the methods of the invention, the first DNA molecules are enzymatically amplified using the polymerase chain reaction. A universal forward primer and a reverse primer are used to prime the polymerase chain reaction. The reverse primer includes a nucleic acid sequence that is selected to specifically hybridize to a portion of a first DNA molecule.
[0039] The reverse primer typically has a length in the range of from 10 nucleotides to 100 nucleotides. In some embodiments, the reverse primer has a length in the range of from 12 nucleotides to 20 nucleotides. The nucleotide sequence of the reverse primer is selected to hybridize to a specific target nucleotide sequence under defined hybridization conditions. The reverse primer and extension primer are both present in the PCR reaction mixture, and so the reverse primer should be sufficiently long so that the melting temperature (Tm) is at least 50° C., but should not be so long that there is extensive overlap with the extension primer which may cause the formation of "primer dimers." "Primer dimers" are formed when the reverse primer hybridizes to the extension primer, and uses the extension primer as a substrate for DNA synthesis, and the extension primer hybridizes to the reverse primer, and uses the reverse primer as a substrate for DNA synthesis. To avoid the formation of "primer dimers," typically the reverse primer and the extension primer are designed so that they do not overlap with each other by more than 6 nucleotides. If it is not possible to make a reverse primer having a Tm of at least 50° C., and wherein the reverse primer and the extension primer do not overlap by more than 6 nucleotides, then it is preferable to lengthen the reverse primer (since Tm usually increases with increasing oligonucleotide length) and decrease the length of the extension primer.
[0040] The reverse primer primes the synthesis of a second DNA molecule that is complementary to the first DNA molecule. The universal forward primer hybridizes to the portion of the second DNA molecule that is complementary to the second portion of the extension primer which is incorporated into all of the first DNA molecules. The universal forward primer primes the synthesis of third DNA molecules. The universal forward primer typically has a length in the range of from 16 nucleotides to 100 nucleotides. In some embodiments, the universal forward primer has a length in the range of from 16 nucleotides to 30 nucleotides. The universal forward primer may include at least one locked nucleic acid molecule. In some embodiments, the universal forward primer includes from 1 to 25 locked nucleic acid molecules. The nucleic acid sequence of an exemplary universal forward primer is set forth in SEQ ID NO:13.
[0041] In general, the greater the number of amplification cycles during the polymerase chain reaction, the greater the amount of amplified DNA that is obtained. On the other hand, too many amplification cycles (e.g., more than 35 amplification cycles) may result in spurious and unintended amplification of non-target double-stranded DNA. Thus, in some embodiments, a desirable number of amplification cycles is between one and 45 amplification cycles, such as from one to 25 amplification cycles, or such as from five to 15 amplification cycles, or such as ten amplification cycles.
[0042] Use of LNA Molecules and Selection of Primer Hybridization Conditions. Hybridization conditions are selected that promote the specific hybridization of a primer molecule to the complementary sequence on a substrate molecule. With respect to the hybridization of a 12 nucleotide first portion of an extension primer to a microRNA, it has been found that specific hybridization occurs at a temperature of 50° C. Similarly, it has been found that hybridization of a 20 nucleotide universal forward primer to a complementary DNA molecule, and hybridization of a reverse primer (having a length in the range of from 12-20 nucleotides, such as from 14-16 nucleotides) to a complementary DNA molecule occurs at a temperature of 50° C. By way of example, it is often desirable to design extension, reverse and universal forward primers that each have a hybridization temperature in the range of from 50° C. to 60° C.
[0043] In some embodiments, LNA molecules can be incorporated into at least one of the extension primer, reverse primer, and universal forward primer to raise the Tm of one, or more, of the foregoing primers to at least 50° C. Incorporation of an LNA molecule into the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may be useful because this portion of the reverse primer is typically no more than 10 nucleotides in length. For example, the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may include at least one locked nucleic acid molecule (e.g., from 1 to 25 locked nucleic acid molecules). In some embodiments, two or three locked nucleic acid molecules are included within the first 8 nucleotides from the 5' end of the reverse primer.
[0044] The number of LNA residues that must be incorporated into a specific primer to raise the Tm to a desired temperature mainly depends on the length of the primer and the nucleotide composition of the primer. A tool for determining the effect on Tm of one or more LNAs in a primer is available on the Internet Web site of Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark.
[0045] Although one or more LNAs can be included in any of the primers used in the practice of the present invention, it has been found that the efficiency of synthesis of cDNA is low if an LNA is incorporated into the extension primer. While not wishing to be bound by theory, LNAs may inhibit the activity of reverse transcriptase.
[0046] Detecting and Measuring the Amount of the Amplified DNA Molecules. The amplified DNA molecules can be detected and quantitated by the presence of detectable marker molecules, such as fluorescent molecules. For example, the amplified DNA molecules can be detected and quantitated by the presence of a dye (e.g., SYBR green) that preferentially or exclusively binds to double stranded DNA during the PCR amplification step of the methods of the present invention. For example, Molecular Probes, Inc. (29851 Willow Creek Road, Eugene, Oreg. 97402) sells quantitative PCR reaction mixtures that include SYBR green dye. By way of further example, another dye (referred to as "BEBO") that can be used to label double stranded DNA produced during real-time PCR is described by Bengtsson, M., et al., Nucleic Acids Research 3/(8):e45 (Apr. 15, 2003), which publication is incorporated herein by reference. Again by way of example, a forward and/or reverse primer that includes a fluorophore and quencher can be used to prime the PCR amplification step of the methods of the present invention. The physical separation of the fluorophore and quencher that occurs after extension of the labeled primer during PCR permits the fluorophore to fluoresce, and the fluorescence can be used to measure the amount of the PCR amplification products. Examples of commercially available primers that include a fluorophore and quencher include Scorpion primers and Uniprimers, which are both sold by Molecular Probes, Inc.
[0047] Representative Uses of the Present Invention. The present invention is useful for producing cDNA molecules from microRNA target molecules. The amount of the DNA molecules can be measured which provides a measurement of the amount of target microRNA molecules in the starting material. For example, the methods of the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in living cells. Again by way of example, the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in different cell types in a living body, thereby producing an "atlas" of the distribution of specific microRNA molecules within the body. Again by way of example, the present invention can be used to measure changes in the amount of specific microRNA molecules (e.g., specific siRNA molecules) in response to a stimulus, such as in response to treatment of a population of living cells with a drug.
[0048] Thus, in another aspect, the present invention provides methods for measuring the amount of a target microRNA in a multiplicity of different cell types within a living organism (e.g., to make a microRNA "atlas" of the organism). The methods of this aspect of the invention each include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method comprising the steps of: (1) using primer extension to make a DNA molecule complementary to the target microRNA molecule isolated from a cell type of a living organism; (2) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules, and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the methods, at least one of the forward primer and the reverse primer comprises at least one locked nucleic acid molecule. The measured amounts of amplified DNA molecules can, for example, be stored in an interrogatable database in electronic form, such as on a computer-readable medium (e.g., a floppy disc).
[0049] In some embodiments, the methods may be used to discriminate between two or more mammalian target microRNA that have a similar sequence in a sample from a living organism, the method comprising the steps of: (a) producing a first DNA molecule that is complementary to the first microRNA molecule using a first extension primer specific to the first microRNA molecule; (b) amplifying the first DNA molecule to produce a first population of amplified DNA molecules using a universal forward primer and a first reverse primer; (c) producing a second DNA molecule that is complementary to the second microRNA molecule using a second extension primer specific to the second microRNA molecule; (d) amplifying the second DNA molecule to produce a second population of amplified DNA molecules using a universal forward primer and a second reverse primer; (e) measuring the amount of the first and second population of amplified DNA molecules, wherein the first and second extension primers or the first and second reverse primers differ by one or more nucleotides in the portion that is complementary to the target microRNA. This method may be used to discriminate between microRNA targets that differ by one, two, three or more nucleotides, by designing the gene-specific region of the first and second extension primers to hybridize to the region of the microRNA targets that are not identical.
[0050] In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.
[0051] In another aspect, the invention provides sets of nucleic acid primers consisting of SEQ ID NO:500 to SEQ ID NO: 965, as shown in TABLE 8. The sets of primer molecules of the invention can be used for the detection of microRNA target molecules from human, mouse, and rat, using the methods of the invention described herein.
[0052] In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer, and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kits, at least one of the universal forward and reverse primers includes at least one locked nucleic acid molecule.
[0053] The extension primer, universal forward and reverse primers for inclusion in the kit may be designed to detect any mammalian target microRNA in accordance with the methods described herein. Nonlimiting examples of human target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 1, TABLE 2, and TABLE 6. Nonlimiting examples of murine target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 7. A nonlimiting example of a universal forward primer is set forth as SEQ ID NO: 13.
[0054] In certain embodiments, the kit includes a set of primers comprising an extension primer, reverse and universal forward primers for a selected target microRNA molecule that each have a hybridization temperature in the range of from 50° C. to 60° C.
[0055] In certain embodiments, the kit includes a plurality of primer sets that may be used to detect a plurality of mammalian microRNA targets, such as two microRNA targets up to several hundred microRNA targets.
[0056] In certain embodiments, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: of miR-1, miR-7, miR-9*, miR-10a, miR-10b, miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-22, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-28, miR-29a, miR-29b, miR-29c, miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-30e-5p, miR-30e-3p, miR-31, miR-32, miR-33, miR-34a, miR-34b, miR-34c, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-99b, miR-100, miR-101, miR-103, miR-105, miR-106a, miR-107, miR-122, miR-122a, miR-124, miR-124, miR-124a, miR-125 a, miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b, miR-129, miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134, miR-135a, miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-146, miR-147, miR-148a, miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-182*, miR-182, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-189, miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR-198, miR-199a*, miR-199a, miR-199b, miR-200a, miR-200b, miR-200c, miR-202, miR-203, miR-204, miR-205, miR-206, miR-208, miR-210, miR-211, miR-212, miR-213, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-220, miR-221, miR-222, miR-223, miR-224, miR-296, miR-299, miR-301, miR-302a*, miR-302a, miR-302b*, miR-302b, miR-302d, miR-302c*, miR-302c, miR-320, miR-323, miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-330, miR-331, miR-337, miR-338, miR-339, miR-340, miR-342, miR-345, miR-346, miR-363, miR-367, miR-368, miR-370, miR-371, miR-372, miR-373*, miR-373, miR-374, miR-375, miR-376b, miR-378, miR-379, miR-380-5p, miR-380-3p, miR-381, miR-382, miR-383, miR-410, miR-412, miR-422a, miR-422b, miR-423, miR-424, miR-425, miR-429, miR-431, miR-448, miR-449, miR-450, miR-451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, let7i, miR-376a, and miR-377. The sequences of the above-mentioned microRNA targets are provided in "the miRBase sequence database" as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/.
[0057] Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.
[0058] In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: miR-1, miR-7, miR-10b, miR-26a, miR-26b, miR-29a, miR-30e-3p, miR-95, miR-107, miR-141, miR-143, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-190, miR-193, miR-194, miR-195, miR-202, miR-206, miR-208, miR-212, miR-221, miR-222, miR-224, miR-296, miR-299, miR-302c*, miR-302c, miR-320, miR-339, miR363, miR-376b, miR379, miR410, miR412, miR424, miR429, miR431, miR449, miR451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, and let7i. Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.
[0059] In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human, mouse or rat microRNA target templates: miR-1, miR-9, miR-18b, miR-20b, miR-92b, miR-146b, miR-181d, miR-193b, miR-194, miR-206, miR-291a-3p, miR-291b-3p, miR-301b, miR-329, miR-346, miR-351, miR-362, miR-362-3p, miR-369-5p, miR-384, miR-409-3p, miR-409-5p, miR-425-5p, miR-449b, miR-455, miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487b, miR-488, miR-489, miR-490, miR-491, miR-493-3p, miR-494, miR-495, miR-497, miR-499, miR-500, miR-501, miR-503, miR-505, miR-519a, miR-519b, miR-519c, miR-519d, miR-520a, miR-520b, miR-520d, miR-520e, miR-520f, miR-532, miR-539, miR-542-3p, miR-542-5p, miR-615, miR-652, miR-668, miR-671, miR-675-5p, miR-699, miR-721, and miR-758.
[0060] Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 8.
[0061] In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 493, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7.
[0062] In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 47, 48, 49, 50, 55, 56, 81, 82, 83, 84, 91, 92, 103, 104, 123, 124, 145, 146, 193, 194, 197, 198, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 239, 240, 247, 248, 253, 254, 255, 256, 257, 258, 277, 278, 285, 286, 287, 288, 293, 294, 301, 302, 309, 310, 311, 312, 315, 316, 317, 318, 319, 320, 333, 334, 335, 336, 337, 338, 359, 360, 369, 370, 389, 390, 393, 394, 405, 406, 407, 408, 415, 416, 419, 420, 421, 422, 425, 426, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 461 and 462, as shown in TABLE 6.
[0063] In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 500 to SEQ ID NO: 965, as shown in TABLE 8.
[0064] A kit of the invention can also provide reagents for primer extension and amplification reactions. For example, in some embodiments, the kit may further include one or more of the following components: a reverse transcriptase enzyme, a DNA polymerase enzyme, a Tris buffer, a potassium salt (e.g., potassium chloride), a magnesium salt (e.g., magnesium chloride), a reducing agent (e.g., dithiothreitol), and deoxynucleoside triphosphates (dNTPs).
[0065] In various embodiments, the kit may include a detection reagent such as SYBR green dye or BEBO dye that preferentially or exclusively binds to double stranded DNA during a PCR amplification step. In other embodiments, the kit may include a forward and/or reverse primer that includes a fluorophore and quencher to measure the amount of the PCR amplification products.
[0066] The kit optionally includes instructions for using the kit in the detection and quantitation of one or more mammalian microRNA targets. The kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling in a high throughput manner.
[0067] The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.
Example 1
[0068] This Example describes a representative method of the invention for producing DNA molecules from microRNA target molecules.
[0069] Primer extension was conducted as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme). The following reaction mixture was prepared on ice: [0070] 1 μl of 10 mM dNTPs [0071] 1 μl of 21.1M extension primer [0072] 1-5 μl of target template [0073] 4 μl of "5×cDNA buffer" [0074] 1 μl of 0.1 M DTT [0075] 1 μl of RNAse OUT [0076] 1 μl of SuperScript III® enzyme [0077] water to 20
[0078] The mixture was incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, then cooled to room temperature and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).
[0079] Real-time PCR was conducted using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. A typical 101.11 PCR reaction mixture contained: [0080] 5 μl of 2×SYBR® green master mix (ABI) [0081] 0.8 μl of 10 μM universal forward primer [0082] 0.8 μl of 10 μM reverse primer [0083] 1.4 μl of water [0084] 2.0 μl of target template (10-fold diluted RT reaction)
[0085] The reaction was monitored through 40 cycles of standard "two cycle" PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products was measured.
[0086] The foregoing method was successfully used in eleven primer extension PCR assays for quantitation of endogenous microRNAs present in a sample of total RNA. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in these 11 assays are shown in TABLE 1.
TABLE-US-00001 TABLE 1 Target Primer Primer SEQ microRNA number Name DNA sequence (5' to 3') ID NO gene-specific extension primers1 humanb let7a 357 let7aP4 CATGATCAGCTGGGCCAAGAAACTATACAACCT 2 human miR-1 337 miR1P5 CATGATCAGCTGGGCCAAGATACATACTTCT 3 human miR-15a 344 miR15aP3 CATGATCAGCTGGGCCAAGACACAAACCATTATG 4 human miR-16 351 miR16P2 CATGATCAGCTGGGCCAAGACGCCAATATTTACGT 5 human miR-21 342 miR21P6 CATGATCAGCTGGGCCAAGATCAACATCAGT 6 human miR-24 350 miR24P5 CATGATCAGCTGGGCCAAGACTGTTCCTGCTG 7 human miR-122 222 122-E5F CATGATCAGCTGGGCCAAGAACAAACACCATTGTCA 8 human miR-124 226 124-E5F CATGATCAGCTGGGCCAAGATGGCATTCACCGCGTG 9 human miR-143 362 miR143P5 CATGATCAGCTGGGCCAAGATGAGCTACAGTG 10 human miR-145 305 miR145P2 CATGATCAGCTGGGCCAAGAAAGGGATTCCTGGGAA 11 human miR-155 367 miR155P3 CATGATCAGCTGGGCCAAGACCCCTATCACGAT 12 universal forward primer 230 E5F CATGATCAGCTGGGCCAAGA 13 RNA species-specific reverse primers2 human let7a 290 miRlet7a- TG+AGGT+AGTAGGTTG 14 1,2,3R human miR-1 285 miR1-1,2R TG+GAA+TG+TAAAGAAGTA 15 human miR-15a 287 miR15aR TAG+CAG+CACATAATG 16 human miR-16 289 miR16-1,2R T+AGC+AGCACGTAAA 17 human miR-21 286 miR21R T+AG+CT+TATCAGACTGAT 18 human miR-24 288 miR24-1,2R TGG+CTCAGTTCAGC 19 human miR-122 234 122LNAR T+G+GAG+TGTGACAA 20 human miR-124 235 124LNAR T+TAA+GGCACGCG 21 human miR-143 291 miR143R TG+AGA+TGAAGCACTG 22 human miR-145 314 miR145R2 GT+CCAGTTTTCCCA 23 human miR-155 293 miR155R T+TAA+TG+CTAATCGTGA 24 1Universal forward primer binding sites are shown in italics. The overlap with the RNA-specific reverse primers are underlined. 2LNA molecules are preceded by a "+". Region of overlap of the reverse primers with the corresponding extension primers are underlined.
[0087] The assay was capable of detecting microRNA in a concentration range of from 2 nM to 20 fM. The assays were linear at least up to a concentration of 2 nM of synthetic microRNA (>1,000,000 copies/cell).
Example 2
[0088] This Example describes the evaluation of the minimum sequence requirements for efficient primer-extension mediated cDNA synthesis using a series of extension primers for microRNA assays having gene specific regions that range in length from 12 to 3 base pairs.
[0089] Primer Extension Reactions. Primer extension was conducted using the target molecules miR-195 and miR-215 as follows. The target templates miR-195 and miR-215 were diluted to 1 nM RNA (100,000 copies/cell) in TE zero plus 100 ng/μl total yeast RNA. A no template control (NTC) was prepared with TE zero plus 100 ng/μl total yeast RNA.
[0090] The reverse transcriptase reactions were carried out as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme) using a series of extension primers for miR-195 (SEQ ID NO: 25-34) and a series of extension primers for miR-215 (SEQ ID NO: 35-44) the sequences of which are shown below in TABLE 2.
[0091] The following reaction mixtures were prepared on ice: [0092] Set 1: No Template Control [0093] 37.5 μl water [0094] 12.5 μl of 10 mM dNTPs [0095] 12.5 μl 0.1 mM DTT [0096] 50 μl of "5×cDNA buffer" [0097] 12.5 μl RNAse OUT [0098] 12.5 μl Superscript III® reverse transcriptase enzyme [0099] 12.5 μl 1 μg/μl Hela cell total RNA (Ambion) [0100] plus 50 μl of 2 μM extension primer [0101] plus 50 μl TEzero+yeast RNA [0102] Set 2: Spike-in Template [0103] 37.5 μl water [0104] 12.5 μl of 10 mM dNTPs [0105] 12.5 μl 0.1 mM DTT [0106] 50 μl of "5×cDNA buffer" [0107] 12.5 μl RNAse OUT [0108] 12.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen) [0109] 12.5 μl 1 μg/μl Hela cell total RNA (Ambion) [0110] plus 50 μl of 2 μM extension primer [0111] plus 50 μl 1 nM RNA target template (miR-195 or miR-215) [0112] serially diluted in 10-fold increments
[0113] The reactions were incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).
[0114] Quantitative Real-Time PCR Reactions. Following reverse transcription, quadruplicate measurements of cDNA were made by quantitative real-time (qPCR) using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. The following reaction mixture was prepared: [0115] 5 μl of 2×SYBR green master mix (ABI) [0116] 0.8 μl of 10 μM universal forward primer (SEQ ID NO: 13) [0117] 0.8 μl of 10 μM reverse primer (miR-195RP:SEQ ID NO: 45 or [0118] miR215RP: SEQ ID NO: 46) [0119] 1.4 μl of water [0120] 2.0 μl of target template (10-fold diluted miR-195 or miR-215 RT reaction)
[0121] Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard "two cycle" PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the miR-195 and miR-215 assays are shown below in TABLE 2. The assay results for miR-195 are shown below in TABLE 3 and the assay results for miR-215 are shown below in TABLE 4.
TABLE-US-00002 TABLE 2 Target Primer SEQ ID microRNA number Primer Name DNA sequence (5' to 3') NO: gene-specific extension primers1 miR-195 646 mir195-GS1 CATGATCAGCTGGGCCAAGAGCCAATATTTCT 25 miR-195 647 mir195-GS2 CATGATCAGCTGGGCCAAGAGCCAATATTTC 26 miR-195 648 mir195-GS3 CATGATCAGCTGGGCCAAGAGCCAATATTT 27 miR-195 649 mir195-GS4 CATGATCAGCTGGGCCAAGAGCCAATATT 28 miR-195 650 mir195-GS5 CATGATCAGCTGGGCCAAGAGCCAATAT 29 miR-195 651 mir195-GS6 CATGATCAGCTGGGCCAAGAGCCAATA 30 miR-195 652 mir195-GS7 CATGATCAGCTGGGCCAAGAGCCAAT 31 miR-195 653 mir195-GS8 CATGATCAGCTGGGCCAAGAGCCAA 32 miR-195 654 mir195-GS9 CATGATCAGCTGGGCCAAGAGCCA 33 miR-195 655 mir195-GS10 CATGATCAGCTGGGCCAAGAGCC 34 miR-215 656 mir215-GS1 CATGATCAGCTGGGCCAAGAGTCTGTCAATTC 35 miR-215 657 mir215-GS2 CATGATCAGCTGGGCCAAGAGTCTGTCAATT 36 miR-215 658 mir215-GS3 CATGATCAGCTGGGCCAAGAGTCTGTCAAT 37 miR-215 659 mir215-GS4 CATGATCAGCTGGGCCAAGAGTCTGTCAA 38 miR-215 660 mir215-GS5 CATGATCAGCTGGGCCAAGAGTCTGTCA 39 miR-215 661 mir215-GS6 CATGATCAGCTGGGCCAAGAGTCTGTC 40 miR-215 662 mir215-GS7 CATGATCAGCTGGGCCAAGAGTCTGT 41 miR-215 663 mir215-GS8 CATGATCAGCTGGGCCAAGAGTCTG 42 miR-215 664 mir215-GS9 CATGATCAGCTGGGCCAAGAGTCT 43 miR-215 665 mir215-GS10 CATGATCAGCTGGGCCAAGAGTC 44 RNA species-specific reverse primers2 miR-195 442 mir195RP T+AGC+AGCACAGAAAT 45 miR-215 446 mir215RP A+T+GA+CCTATGAATTG 46 1Universal forward primer binding sites are shown in italics. 2The "+" symbol precedes the LNA molecules.
[0122] Results:
[0123] The sensitivity of each assay was measured by the cycle threshold (Ct) value which is defined as the cycle count at which fluorescence was detected in an assay containing microRNA target template. The lower this Ct value (e.g. the fewer number of cycles), the more sensitive was the assay. For microRNA samples, it was generally observed that while samples that contain template and no template controls both eventually cross the detection threshold, the samples with template do so at a much lower cycle number. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) and broad dynamic range (ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.
[0124] The results of the miR195 and miR215 assays using extension primers having a gene specific portion ranging in size from 12 nucleotides to 3 nucleotides are shown below in TABLE 3 and TABLE 4, respectively. The results of these experiments unexpectedly demonstrate that gene-specific priming sequences as short as 3 nucleotides exhibit template specific priming. For both the miR-195 assay sets (shown in TABLE 3) and the miR-215 assay sets (shown in TABLE 4), the results demonstrate that the dynamic range (ΔCt) for both sets of assays are fairly consistent for extension primers having gene specific regions that are greater or equal to 8 nucleotides in length. The dynamic range of the assay (ΔCt) begins to decrease for extension primers having gene specific regions below 8 nucleotides, with a reduction in assay specificity below 7 nucleotides in the miR-195 assays, and below 6 nucleotides in the miR-215 assays. A melting point analysis of the miR-215 samples demonstrated that even at 3 nucleotides, there is specific PCR product present in the plus template samples (data not shown). Taken together, these data demonstrate that the gene specific region of extension primers is ideally ≧8 nucleotides, but can be as short as 3 nucleotides in length.
TABLE-US-00003 TABLE 3 MIR195 ASSAY RESULTS GS Primer Ct: No Ct: Plus Length Template Control Template Δ Ct 12 34.83 20.00 14.82 12 34.19 19.9 14.3 11 40.0 19.8 20.2 10 36.45 21.2 15.2 9 36.40 22.2 14.2 8 40.0 23.73 16.27 7 36.70 25.96 10.73 6 30.95 26.58 4.37 5 30.98 31.71 -0.732 4 32.92 33.28 -0.364 3 35.98 35.38 -0.605 Ct = the cycle count where the fluorescence exceeds the threshold of detection. Δ Ct = the difference between the Ct value with template and no template.
TABLE-US-00004 TABLE 4 MIR215 ASSAY RESULTS GS Primer Ct: No Ct: Plus Length Template Control Template Δ Ct 12 33.4 13.57 19.83 12 33.93 14.15 19.77 11 35.51 15.76 19.75 10 35.33 15.49 19.84 9 36.02 16.84 19.18 8 35.79 17.07 18.72 7 32.29 17.58 14.71 6 34.38 20.62 13.75 5 34.41 28.65 5.75 4 36.36 33.92 2.44 3 35.09 33.38 1.70 Ct = the cycle count where the fluorescence exceeds the threshold of detection. Δ Ct = the difference between the Ct value with template and no template.
Example 3
[0125] This Example describes assays and primer sets designed for quantitative analysis of human microRNA expression patterns.
[0126] Primer Design:
[0127] microRNA target templates: the sequence of the target templates as described herein are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute Web site in the "miRBase sequence database" as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.
[0128] Extension primers: gene specific primers for primer extension of a microRNA to form a cDNA followed by quantitative PCR (qPCR) amplification were designed to (1) convert the RNA template into cDNA; (2) to introduce a "universal" PCR binding site (SEQ ID NO:1) to one end of the cDNA molecule; and (3) to extend the length of the cDNA to facilitate subsequent monitoring by qPCR.
[0129] Reverse primers: unmodified reverse primers and locked nucleic acid (LNA) containing reverse primers (RP) were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). For the locked nucleic acid containing reverse primers, two or three LNA modified bases were substituted within the first 8 nucleotides from the 5' end of the reverse primer oligonucleotide, as shown below in the exemplary reverse primer sequences provided in TABLE 6. The LNA base substitutions were selected to raise the predicted Tm of the primer by the highest amount, and the final predicted Tm of the selected primers were specified to be preferably less than or equal to 55° C.
[0130] An example describing an assay utilizing an exemplary set of primers the detection of miR-95 and miR-424 is described below.
[0131] Primer Extension Reactions: primer extension was conducted using DNA templates corresponding to miR-95 and miR-424 as follows. The DNA templates were diluted to 0 nM, 1 nM, 100 pM, 10 pM, and 1 pM dilutions in TE zero (10 mM Tris pH 7.6, 0.1 mM EDTA) plus 100 ng/μl yeast total RNA (Ambion, Austin, Tex.).
[0132] The reverse transcriptase reactions were carried out using the following primers:
[0133] Extension primers: (diluted to 500 nM)
TABLE-US-00005 miR-95GSP CATGATCAGCTGGGCCAAGATGCTCAATAA (SEQ ID NO: 123) miR-424GSP CATGATCAGCTGGGCCAAGATTCAAAACAT (SEQ ID NO: 415)
[0134] Reverse primers: (diluted to 10 mM)
TABLE-US-00006 miR-95_RP4 TT+CAAC+GGGTATTTATTGA (SEQ ID NO: 124) miR-424RP2 C+AG+CAGCAATTCATGTTTT (SEQ ID NO: 416)
[0135] Reverse Transcription (per reaction):
[0136] 2 μl water
[0137] 2 μl of "5×cDNA buffer" (InVitrogen, Carlsbad, Calif.)
[0138] 0.5 μl of 0.1 mM DTT (InVitrogen, Carlsbad, Calif.)
[0139] 0.5 μl of 10 mM dNTPs (InVitrogen, Carlsbad, Calif.)
[0140] 0.5 μl RNAse OUT (InVitrogen, Carlsbad, Calif.)
[0141] 0.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen, Carlsbad, Calif.)
[0142] 2 μl of extension primer plus 2 μl of template dilution
[0143] The reactions were mixed and incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE zero.
[0144] Quantitative Real-Time PCR Reactions (per reaction): [0145] 5 μl 12×SYBR mix (Applied Biosystems, Foster City, Calif.) [0146] 1.4 μl water [0147] 0.8 μl universal primer (CATGATCAGCTGGGCCAAGA (SEQ ID NO: 13)) [0148] 2.0 μl of diluted reverse transcription (RT) product from above.
[0149] Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard "two cycle" PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the representative miR-95 and miR-424 assays as well as primer sets for 212 different human microRNA templates are shown below in TABLE 6. Primer sets for assays requiring extensive testing and design modification to achieve a sensitive assay with a high dynamic range are indicated in TABLE 6 with the symbol # following the primer name.
[0150] Results:
[0151] TABLE 5 shows the Ct values (averaged from four samples) from the miR-95 and miR-424 assays, which are plotted in the graph shown in FIG. 2. The results of these assays are provided as representative examples in order to explain the significance of the assay parameters shown in TABLE 6 designated as slope (column 6), intercept (column 7) and background (column 8).
[0152] As shown in TABLE 5, the Ct value for each template at various concentrations is provided. The Ct values (x-axis) are plotted as a function of template concentration (y-axis) to generate a standard curve for each assay, as shown in FIG. 2. The slope and intercept define the assay measurement characteristics that permit an estimation of number of copies/cell for each microRNA. For example, when the Ct values for 50 μg total RNA input for the miR-95 assay are plotted, a standard curve is generated with a slope and intercept of -0.03569 and 9.655, respectively. When these standard curve parameters are applied to the Ct of an unknown sample (x), they yield log 10 (copies/20 pg total RNA) (y). Because the average cell yields 20 pg of total RNA, these measurements equate to copies of microRNA/cell. The background provides an estimate of the minimum copy number that can be measured in a sample and is computed by inserting the no template control (NTC) value into this equation. In this example, as shown in TABLE 6, miR-95 yields a background of 1.68 copies/20 pg at 50 μg of RNA input.
[0153] As further shown in TABLE 6, reverse primers that do not contain LNA may also be used in accordance with the methods of the invention. See, e.g., SEQ ID NO:494-499. The sensitivity and dynamic range of the assays using non-LNA containing reverse primers SEQ ID NO:494-499, yielded similar results to the corresponding assays using LNA-containing reverse primers.
TABLE-US-00007 TABLE 5 Ct Values (averaged from four samples) Template concentration 10 nM 1 nM 0.1 nM 0.01 nM 0.001 nM NTC copies/20 pg .sup. 500,000 50,000 .sup. 5000 500 50 RNA (50 μg input) copies/20 pg 5,000,000 500,000 50,000 5000 500 RNA (5 μg input) miR-95 11.71572163 14.17978 17.46353 19.97259 23.33171 27.44383 miR-424 10.47708975 12.76806 15.69251 18.53729 21.56897 23.2813 log10 (copies 5.698970004 4.69897 3.69897 2.69897 1.69897 for 50 μg input)
TABLE-US-00008 TABLE 6 PRIMERS TO DETECT HUMAN MICRORNA TARGET TEMPLATES Human Target Background micro RNA input RNA Extension Primer Name Extension Primer Sequence Reverse Primer Name Reverse Primer Sequence Slope Intercept 50 ug 5 ug miR-1 miR1GSP10# CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP# T+G+GAA+TG+TAAAGAAGT -0.2758 8.3225 2.44 24.36 SEQ ID NO: 47 SEQ ID NO: 48 miR-7 miR-7GSP10# CATGATCAGCTGGGCCAAGACAACAAAATC miR-7_RP6# T+GGAA+GACTAGTGATTTT -0.2982 10.435 11.70 116.99 SEQ ID NO: 49 SEQ ID NO: 50 miR-9* miR-9*GSP CATGATCAGCTGGGCCAAGAACTTTCGGTT miR-9*RP TAAA+GCT+AGATAACCG -0.2405 8.9145 3.71 37.15 SEQ ID NO: 51 SEQ ID NO: 52 miR-10a miR-10aGSP CATGATCAGCTGGGCCAAGACACAAATTCG miR-10aRP T+AC+CCTGTAGATCCG -0.2755 8.6976 0.09 0.94 SEQ ID NO: 53 SEQ ID NO: 54 miR-10b miR- CATGATCAGCTGGGCCAAGAACAAATTCGGT miR- TA+CCC+TGT+AGAACCGA -0.3505 8.7109 0.55 5.52 10b_GSP11# SEQ ID NO: 55 10b_RP2# SEQ ID NO: 56 miR-15a miR-15aGSP CATGATCAGCTGGGCCAAGACACAAACCAT miR-15aRP T+AG+CAGCACATAATG -0.2831 8.4519 4.40 44.01 SEQ ID NO: 57 SEQ ID NO: 58 miR-15b miR-15bGSP2 CATGATCAGCTGGGCCAAGATGTAAACCA miR-15bRP T+AG+CAGCACATCAT -0.2903 8.4206 0.18 1.84 SEQ ID NO: 59 SEQ ID NO: 60 miR-16 miR-16GSP2 CATGATCAGCTGGGCCAAGACGCCAATAT miR-16RP T+AG+CAGCACGTAAA -0.2542 9.3689 1.64 16.42 SEQ ID NO: 61 SEQ ID NO: 62 miR-17- miR-17-3pGSP CATGATCAGCTGGGCCAAGAACAAGTGCCT miR-17-3pRP A+CT+GCAGTGAAGGC -0.2972 8.2625 1.08 10.78 3p SEQ ID NO: 63 SEQ ID NO: 64 miR-17- miR-17- CATGATCAGCTGGGCCAAGAACTACCTGC miR-17-5pRP C+AA+AGTGCTTACAGTG -0.2956 7.9101 0.13 1.32 5p 5pGSP2 SEQ ID NO: 65 SEQ ID NO: 66 miR-19a miR-19aGSP2 CATGATCAGCTGGGCCAAGATCAGTTTTG miR-19aRP TG+TG+CAAATCTATGC -0.2984 9.461 0.02 0.23 SEQ ID NO: 67 SEQ ID NO: 68 miR-19b miR-19bGSP CATGATCAGCTGGGCCAAGATCAGTTTTGC miR-19bRP TG+TG+CAAATCCATG -0.294 8.1434 2.26 22.55 SEQ ID NO: 69 SEQ ID NO: 70 miR-20 miR-20GSP3 CATGATCAGCTGGGCCAAGACTACCTGC miR-20RP T+AA+AGTGCTTATAGTGCA -0.2979 7.9929 0.16 1.60 SEQ ID NO: 71 SEQ ID NO: 72 miR-21 miR-21GSP2 CATGATCAGCTGGGCCAAGATCAACATCA miR-21RP T+AG+CTTATCAGACTGATG -0.2849 8.1624 1.80 17.99 SEQ ID NO: 73 SEQ ID NO: 74 miR-23a miR-23aGSP CATGATCAGCTGGGCCAAGAGGAAATCCCT miR-23aRP A+TC+ACATTGCCAGG -0.3172 9.4253 2.41 24.08 SEQ ID NO: 75 SEQ ID NO: 76 miR-23b miR-23bGSP CATGATCAGCTGGGCCAAGAGGTAATCCCT miR-23bRP A+TC+ACATTGCCAGG -0.2944 9.0985 5.39 53.85 SEQ ID NO: 77 SEQ ID NO: 78 miR-25 miR-25GSP CATGATCAGCTGGGCCAAGATCAGACCGAG miR-25RP C+AT+TGCACTTGTCTC -0.3009 8.2482 1.52 15.19 SEQ ID NO: 79 SEQ ID NO: 80 miR-26a miR-26aGSP9# CATGATCAGCTGGGCCAAGAGCCTATCCT miR- TT+CA+AGTAATCCAGGAT -0.2807 8.558 0.26 2.56 SEQ ID NO: 81 26aRP2# SEQ ID NO: 82 miR-26b miR-26bGSP9# CATGATCAGCTGGGCCAAGAAACCTATCC miR- TT+CA+AGT+AATTCAGGAT -0.2831 8.7885 0.37 3.67 SEQ ID NO: 83 26bRP2# SEQ ID NO: 84 miR-27a miR-27aGSP CATGATCAGCTGGGCCAAGAGCGGAACTTA miR-27aRP TT+CA+CAGTGGCTAA -0.2765 9.5239 5.15 51.51 SEQ ID NO: 85 SEQ ID NO: 86 miR-27b miR-27bGSP CATGATCAGCTGGGCCAAGAGCAGAACTTA miR-27bRP TT+CA+CAGTGGCTAA -0.28 9.5483 5.97 59.71 SEQ ID NO: 87 SEQ ID NO: 88 miR-28 miR-28GSP CATGATCAGCTGGGCCAAGACTCAATAGAC miR-28RP A+AG+GAGCTCACAGT -0.3226 10.071 7.19 71.87 SEQ ID NO: 89 SEQ ID NO: 90 miR-29a miR-29aGSP8# CATGATCAGCTGGGCCAAGAAACCGATT miR- T+AG+CACCATCTGAAAT -0.29 8.8731 0.04 0.38 SEQ ID NO: 91 29aRP2# SEQ ID NO: 92 miR-29b miR-29bGSP2 CATGATCAGCTGGGCCAAGAAACACTGAT miR-29bRP2 T+AG+CACCATTTGAAATCAG -0.3162 9.6276 3.56 35.57 SEQ ID NO: 93 SEQ ID NO: 94 miR-30a- miR-30a- CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30a- T+GT+AAACATCCTCGAC -0.2772 9.0694 1.92 19.16 5p 5pGSP SEQ ID NO: 95 5pRP SEQ ID NO: 96 miR-30b miR-30bGSP CATGATCAGCTGGGCCAAGAAGCTGAGTGT miR-30bRP TGT+AAA+CATCCTACACT -0.2621 8.5974 0.11 1.13 SEQ ID NO: 97 SEQ ID NO: 98 miR-30c miR-30cGSP CATGATCAGCTGGGCCAAGAGCTGAGAGTG miR-30cRP TGT+AAA+CATCCTACACT -0.2703 8.699 0.15 1.48 SEQ ID NO: 99 SEQ ID NO: 100 miR-30d miR-30dGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30dRP T+GTAAA+CATCCCCG -0.2506 9.3875 0.23 2.31 SEQ ID NO: 101 SEQ ID NO: 102 miR-30e- miR-30e- CATGATCAGCTGGGCCAAGAGCTGTAAAC miR-30e- CTTT+CAGT+CGGATGTTT -0.325 11.144 6.37 63.70 3p 3pGSP9# SEQ ID NO: 103 3pRP5# SEQ ID NO: 104 miR-30e- miR-30e- CATGATCAGCTGGGCCAAGATCCAGTCAAG miR-30e- TG+TAAA+CATCCTTGAC -0.2732 8.1604 8.50 85.03 5p 5pGSP SEQ ID NO: 105 5pRP SEQ ID NO: 106 miR-31 miR-31GSP CATGATCAGCTGGGCCAAGACAGCTATGCC miR-31RP G+GC+AAGATGCTGGC -0.3068 8.2605 3.74 37.43 SEQ ID NO: 107 SEQ ID NO: 108 miR-32 miR-32GSP CATGATCAGCTGGGCCAAGAGCAACTTAGT miR-32RP TATTG+CA+CATTACTAAG -0.2785 8.9581 0.39 3.93 SEQ ID NO: 109 SEQ ID NO: 110 miR-33 miR-33GSP2 CATGATCAGCTGGGCCAAGACAATGCAAC miR-33RP G+TG+CATTGTAGTTGC -0.3031 8.42 2.81 28.14 SEQ ID NO: 111 SEQ ID NO: 112 miR-34a miR-34aGSP CATGATCAGCTGGGCCAAGAAACAACCAGC miR-34aRP T+GG+CAGTGTCTTAG -0.3062 9.1522 2.40 23.99 SEQ ID NO: 113 SEQ ID NO: 114 miR-34b miR-34bGSP CATGATCAGCTGGGCCAAGACAATCAGCTA miR-34bRP TA+GG+CAGTGTCATT -0.3208 9.054 0.04 0.37 SEQ ID NO: 115 SEQ ID NO: 116 miR-34c miR-34cGSP CATGATCAGCTGGGCCAAGAGCAATCAGCT miR-34cRP A+GG+CAGTGTAGTTA -0.2995 10.14 1.08 10.83 SEQ ID NO: 117 SEQ ID NO: 118 miR-92 miR-92GSP CATGATCAGCTGGGCCAAGACAGGCCGGGA miR-92RP T+AT+TGCACTTGTCCC -0.3012 8.6908 8.92 89.17 SEQ ID NO: 119 SEQ ID NO: 120 miR-93 miR-93GSP CATGATCAGCTGGGCCAAGACTACCTGCAC miR-93RP AA+AG+TGCTGTTCGT -0.3025 7.9933 4.63 46.30 SEQ ID NO: 121 SEQ ID NO: 122 miR-95 miR-95GSP# CATGATCAGCTGGGCCAAGATGCTCAATAA miR- TT+CAAC+GGGTATTTATTGA -0.3436 9.655 1.68 16.80 SEQ ID NO: 123 95_RP4# SEQ ID NO: 124 miR-96 miR-96GSP CATGATCAGCTGGGCCAAGAGCAAAAATGT miR-96RP T+TT+GGCACTAGCAC -0.2968 9.2611 0.00 0.05 SEQ ID NO: 125 SEQ ID NO: 126 miR-98 miR-98GSP CATGATCAGCTGGGCCAAGAAACAATACAA miR-98RP TGA+GGT+AGTAAGTTG -0.2797 9.5654 1.05 10.48 SEQ ID NO: 127 SEQ ID NO: 128 miR-99a miR-99aGSP CATGATCAGCTGGGCCAAGACACAAGATCG miR-99aRP A+AC+CCGTAGATCCG -0.2768 8.781 0.21 2.08 SEQ ID NO: 129 SEQ ID NO: 130 miR-99b miR-99bGSP CATGATCAGCTGGGCCAAGACGCAAGGTCG miR-99bRP C+AC+CCGTAGAACCG -0.2747 7.9855 0.25 2.53 SEQ ID NO: 131 SEQ ID NO: 132 miR-100 miR-100GSP CATGATCAGCTGGGCCAAGACACAAGTTCG miR-100RP A+AC+CCGTAGATCCG -0.2902 8.669 0.04 0.35 SEQ ID NO: 133 SEQ ID NO: 134 miR-101 miR-101GSP CATGATCAGCTGGGCCAAGACTTCAGTTAT miR-101RP TA+CAG+TACTGTGATAACT -0.3023 8.2976 0.46 4.63 SEQ ID NO: 135 SEQ ID NO: 136 miR-103 miR-103GSP CATGATCAGCTGGGCCAAGATCATAGCCCT miR-103RP A+GC+AGCATTGTACA -0.3107 8.5776 0.02 0.21 SEQ ID NO: 137 SEQ ID NO: 138 miR-105 miR-105GSP CATGATCAGCTGGGCCAAGAACAGGAGTCT miR-105RP T+CAAA+TGCTCAGACT -0.2667 8.9832 0.93 9.28 SEQ ID NO: 139 SEQ ID NO: 140 miR-106a miR-106aGSP CATGATCAGCTGGGCCAAGAGCTACCTGCA miR-106aRP AAA+AG+TGCTTACAGTG -0.3107 8.358 0.03 0.31 SEQ ID NO: 141 SEQ ID NO: 142 miR-106b miR-106bGSP CATGATCAGCTGGGCCAAGAATCTGCACTG miR-106bRP T+AAAG+TGCTGACAGT -0.2978 8.7838 0.10 1.04 SEQ ID NO: 143 SEQ ID NO: 144 miR-107 miR-107GSP8# CATGATCAGCTGGGCCAAGATGATAGCC miR- A+GC+AGCATTGTACAG -0.304 9.1666 0.34 3.41 SEQ ID NO: 145 107RP2# SEQ ID NO: 146 miR-122a miR-122aGSP CATGATCAGCTGGGCCAAGAACAAACACCA miR-122aRP T+GG+AGTGTGACAAT -0.3016 8.1479 0.06 0.58 SEQ ID NO: 147 SEQ ID NO: 148 miR-124a miR-124aGSP CATGATCAGCTGGGCCAAGATGGCATTCAC miR-124aRP T+TA+AGGCACGCGGT -0.3013 8.6906 0.56 5.63 SEQ ID NO: 149 SEQ ID NO: 150 miR-125a miR-125aGSP CATGATCAGCTGGGCCAAGACACAGGTTAA miR-125aRP T+CC+CTGAGACCCTT -0.2938 8.6754 0.09 0.91 SEQ ID NO: 151 SEQ ID NO: 152 miR-125b miR-125bGSP CATGATCAGCTGGGCCAAGATCACAAGTTA miR-125bRP T+CC+CTGAGACCCTA -0.283 8.1251 0.20 1.99 SEQ ID NO: 153 SEQ ID NO: 154 miR-126 miR-126GSP CATGATCAGCTGGGCCAAGAGCATTATTAC miR-126RP T+CG+TACCGTGAGTA -0.26 8.937 0.18 1.80 SEQ ID NO: 155 SEQ ID NO: 156 miR-126* miR-126*GSP3 CATGATCAGCTGGGCCAAGACGCGTACC miR-126*RP C+ATT+ATTA+CTTTTGGTACG -0.2969 8.184 3.58 35.78 SEQ ID NO: 157 SEQ ID NO: 158 miR-127 miR-127GSP CATGATCAGCTGGGCCAAGAAGCCAAGCTC miR-127RP T+CG+GATCCGTCTGA -0.2432 9.1013 1.11 11.13 SEQ ID NO: 159 SEQ ID NO: 160 miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR-128aRP T+CA+CAGTGAACCGG -0.2866 8.0867 0.16 1.60 SEQ ID NO: 161 SEQ ID NO: 162 miR-128b miR-128bGSP CATGATCAGCTGGGCCAAGAGAAAGAGACC miR-128bRP T+CA+CAGTGAACCGG -0.2923 8.0608 0.07 0.74 SEQ ID NO: 163 SEQ ID NO: 164 miR-129 miR-129GSP CATGATCAGCTGGGCCAAGAGCAAGCCCAG miR-129RP CTTTT+TG+CGGTCTG -0.2942 9.7731 0.88 8.85 SEQ ID NO: 165 SEQ ID NO: 166 miR-130a miR-130aGSP CATGATCAGCTGGGCCAAGAATGCCCTTTT miR-130aRP
C+AG+TGCAATGTTAAAAG -0.2943 8.7465 1.28 12.78 SEQ ID NO: 167 SEQ ID NO: 168 miR-130b miR-130bGSP CATGATCAGCTGGGCCAAGAATGCCCTTTC miR-130bRP C+AG+TGCAATGATGA -0.2377 9.1403 3.14 31.44 SEQ ID NO: 169 SEQ ID NO: 170 miR-132 miR-132GSP CATGATCAGCTGGGCCAAGACGACCATGGC miR-132RP T+AA+CAGTCTACAGCC -0.2948 8.1167 0.11 1.13 SEQ ID NO: 171 SEQ ID NO: 172 miR-133a miR-133aGSP CATGATCAGCTGGGCCAAGAACAGCTGGTT miR-133aRP T+TG+GTCCCCTTCAA -0.295 9.3679 0.10 1.04 SEQ ID NO: 173 SEQ ID NO: 174 miR-133b miR-133bGSP CATGATCAGCTGGGCCAAGATAGCTGGTTG miR-133bRP T+TG+GTCCCCTTCAA -0.3062 8.3649 0.02 0.18 SEQ ID NO: 175 SEQ ID NO: 176 miR-134 miR-134GSP CATGATCAGCTGGGCCAAGACCCTCTGGTC miR-134RP T+GT+GACTGGTTGAC -0.2965 9.0483 0.14 1.39 SEQ ID NO: 177 SEQ ID NO: 178 miR-135a miR-135aGSP CATGATCAGCTGGGCCAAGATCACATAGGA miR-135aRP T+AT+GGCTTTTTATTCCT -0.2914 8.092 1.75 17.50 SEQ ID NO: 179 SEQ ID NO: 180 miR-135b miR-135bGSP CATGATCAGCTGGGCCAAGACACATAGGAA miR-135bRP T+AT+GGCTTTTCATTCC -0.2962 7.8986 0.05 0.49 SEQ ID NO: 181 SEQ ID NO: 182 miR-136 miR-136GSP CATGATCAGCTGGGCCAAGATCCATCATCA miR-136RP A+CT+CCATTTGTTTTGATG -0.3616 10.229 0.68 6.77 SEQ ID NO: 183 SEQ ID NO: 184 miR-137 miR-137GSP CATGATCAGCTGGGCCAAGACTACGCGTAT miR-137RP T+AT+TGCTTAAGAATACGC -0.2876 8.234 8.57 85.71 SEQ ID NO: 185 SEQ ID NO: 186 miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR-138RP A+GC+TGGTGTTGTGA -0.3023 9.0814 0.22 2.19 SEQ ID NO: 187 SEQ ID NO: 188 miR-139 miR-139GSP CATGATCAGCTGGGCCAAGAAGACACGTGC miR-139RP T+CT+ACAGTGCACGT -0.2983 8.1141 6.92 69.21 SEQ ID NO: 189 SEQ ID NO: 190 miR-140 miR-140GSP CATGATCAGCTGGGCCAAGACTACCATAGG miR-140RP A+GT+GGTTTTACCCT -0.2312 8.3231 0.13 1.34 SEQ ID NO: 191 SEQ ID NO: 192 miR-141 miR-141GSP9# CATGATCAGCTGGGCCAAGACCATCTTTA miR- TAA+CAC+TGTCTGGTAA -0.2805 9.6671 0.13 1.26 SEQ ID NO: 193 141RP2# SEQ ID NO: 194 miR-142- miR-142- CATGATCAGCTGGGCCAAGATCCATAAA miR-142- TGT+AG+TGTTTCCTACT -0.2976 8.4046 0.03 0.27 3p 3pGSP3 SEQ ID NO: 195 3pRP SEQ ID NO: 196 miR-143 miR-143GSP8# CATGATCAGCTGGGCCAAGATGAGCTAC miR- T+GA+GATGAAGCACTG -0.3008 9.2675 0.37 3.71 SEQ ID NO: 197 143RP2# SEQ ID NO: 198 miR-144 miR-144GSP2 CATGATCAGCTGGGCCAAGACTAGTACAT miR-144RP TA+CA+GTAT+AGATGATG -0.2407 9.4441 0.95 9.52 SEQ ID NO: 199 SEQ ID NO: 200 miR-145 miR-145GSP2 CATGATCAGCTGGGCCAAGAAAGGGATTC miR-145RP G+TC+CAGTTTTCCCA -0.2937 8.0791 0.39 3.86 SEQ ID NO: 201 SEQ ID NO: 202 miR-146 miR-146GSP3 CATGATCAGCTGGGCCAAGAAACCCATG miR-146RP T+GA+GAACTGAATTCCA -0.2861 8.8246 0.08 0.75 SEQ ID NO: 203 SEQ ID NO: 204 miR-147 miR-147GSP CATGATCAGCTGGGCCAAGAGCAGAAGCAT miR-147RP G+TG+TGTGGAAATGC -0.2989 8.8866 1.65 16.47 SEQ ID NO: 205 SEQ ID NO: 206 miR-148a miR-148aGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR- T+CA+GTGCACTACAGAACT -0.2928 9.4654 1.27 12.65 SEQ ID NO: 207 148aRP2 SEQ ID NO: 208 miR-148b miR-148bGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148bRP T+CA+GTGCATCACAG -0.2982 10.417 0.24 2.44 SEQ ID NO: 209 SEQ ID NO: 210 miR-149 miR-149GSP2 CATGATCAGCTGGGCCAAGAGGAGTGAAG miR-149RP T+CT+GGCTCCGTGTC -0.2996 8.3392 2.15 21.50 SEQ ID NO: 211 SEQ ID NO: 212 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR-150RP T+CT+CCCAACCCTTG -0.2943 8.3945 0.06 0.56 SEQ ID NO: 213 SEQ ID NO: 214 miR-151 miR-151GSP2 CATGATCAGCTGGGCCAAGACCTCAAGGA miR-151RP A+CT+AGACTGAAGCTC -0.2975 8.651 0.16 1.60 SEQ ID NO: 215 SEQ ID NO: 216 miR-152 miR-152GSP2 CATGATCAGCTGGGCCAAGACCCAAGTTC miR-152RP T+CA+GTGCATGACAG -0.2741 8.7404 0.33 3.25 SEQ ID NO: 217 SEQ ID NO: 218 miR-153 miR-153GSP2 CATGATCAGCTGGGCCAAGATCACTTTTG miR-153RP TTG+CAT+AGTCACAAAA -0.2723 9.5732 3.32 33.19 SEQ ID NO: 219 SEQ ID NO: 220 miR-154* miR- CATGATCAGCTGGGCCAAGAAATAGGTCA miR- AATCA+TA+CACGGTTGAC -0.3056 8.8502 0.07 0.74 154*GSP9# SEQ ID NO: 221 154*RP2# SEQ ID NO: 222 miR-154 miR-154GSP9# CATGATCAGCTGGGCCAAGACGAAGGCAA miR- TA+GGTTA+TCCGTGTT -0.3062 9.3947 0.10 0.96 SEQ ID NO: 223 154RP3# SEQ ID NO: 224 miR-155 miR-155GSP8# CATGATCAGCTGGGCCAAGACCCCTATC miR- TT+AA+TGCTAATCGTGATAGG -0.3201 8.474 5.49 54.91 SEQ ID NO: 225 155RP2# SEQ ID NO: 226 miR-181a miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AA+CATT+CAACGCTGTC -0.2919 7.968 1.70 17.05 181aGSP9# SEQ ID NO: 227 181aRP2# SEQ ID NO: 228 miR-181c miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AA+CATT+CAACCTGTCG -0.3102 7.9029 1.08 10.78 181cGSP9# SEQ ID NO: 229 181cRP2# SEQ ID NO: 230 miR-182* miR-182*GSP CATGATCAGCTGGGCCAAGATAGTTGGCAA miR-182*RP T+GG+TTCTAGACTTGC -0.2978 8.5876 4.25 42.47 SEQ ID NO: 231 SEQ ID NO: 232 miR-182 miR-182GSP2 CATGATCAGCTGGGCCAAGATGTGAGTTC miR-182RP TTT+GG+CAATGGTAG -0.2863 9.0854 1.52 15.20 SEQ ID NO: 233 SEQ ID NO: 234 miR-183 miR-183GSP2 CATGATCAGCTGGGCCAAGACAGTGAATT miR-183RP T+AT+GGCACTGGTAG -0.2774 9.9254 1.95 19.51 SEQ ID NO: 235 SEQ ID NO: 236 miR-184 miR-184GSP2 CATGATCAGCTGGGCCAAGAACCCTTATC miR-184RP T+GG+ACGGAGAACTG -0.2906 7.9585 0.05 0.49 SEQ ID NO: 237 SEQ ID NO: 238 miR-186 miR-186GSP9# CATGATCAGCTGGGCCAAGAAAGCCCAAA miR- CA+AA+GAATT+CTCCTTTTGG -0.2861 8.6152 0.32 3.18 SEQ ID NO: 239 186RP3# SEQ ID NO: 240 miR-187 miR-187GSP CATGATCAGCTGGGCCAAGACGGCTGCAAC miR-187RP T+CG+TGTCTTGTGTT -0.2953 7.9329 1.23 12.31 SEQ ID NO: 241 SEQ ID NO: 242 miR-188 miR-188GSP CATGATCAGCTGGGCCAAGAACCCTCCACC miR-188RP C+AT+CCCTTGCATGG -0.2925 8.0782 8.49 84.92 SEQ ID NO: 243 SEQ ID NO: 244 miR-189 miR-189GSP2 CATGATCAGCTGGGCCAAGAACTGATATC miR-189RP G+TG+CCTACTGAGCT -0.2981 8.8964 0.21 2.08 SEQ ID NO: 245 SEQ ID NO: 246 miR-190 miR-190GSP9# CATGATCAGCTGGGCCAAGAACCTAATAT miR- T+GA+TA+TGTTTGATATATT -0.3317 9.8766 0.43 4.34 SEQ ID NO: 247 190RP4# AG SEQ ID NO: 248 miR-191 miR-191GSP2 CATGATCAGCTGGGCCAAGAAGCTGCTTT miR-191RP2 C+AA+CGGAATCCCAAAAG -0.299 9.0317 0.41 4.07 SEQ ID NO: 249 SEQ ID NO: 250 miR-192 miR-192GSP2 CATGATCAGCTGGGCCAAGAGGCTGTCAA miR-192RP C+TGA+CCTATGAATTGAC -0.2924 9.5012 1.10 10.98 SEQ ID NO: 251 SEQ ID NO: 252 miR-193 miR-193GSP9# CATGATCAGCTGGGCCAAGACTGGGACTT miR- AA+CT+GGCCTACAAAG -0.3183 8.9942 0.17 1.72 SEQ ID NO: 253 193RP2# SEQ ID NO: 254 miR-194 mir194GSP8# CATGATCAGCTGGGCCAAGATCCACATG mir194RP# TG+TAA+CAGCAACTCCA -0.3078 8.8045 0.37 3.69 SEQ ID NO: 255 SEQ ID NO: 256 miR-195 miR-195GSP9# CATGATCAGCTGGGCCAAGAGCCAATATT miR- T+AG+CAG+CACAGAAATA -0.2955 10.213 0.76 7.58 SEQ ID NO: 257 195RP3# SEQ ID NO: 258 miR-196b miR-196bGSP CATGATCAGCTGGGCCAAGACCAACAACAG miR-196bRP TA+GGT+AGTTTCCTGT -0.301 8.1641 1.47 14.66 SEQ ID NO: 259 SEQ ID NO: 260 miR-196a miR-196aGSP CATGATCAGCTGGGCCAAGACCAACAACAT miR-196aRP TA+GG+TAGTTTCATGTTG -0.2932 8.0448 8.04 80.37 SEQ ID NO: 261 SEQ ID NO: 262 miR-197 miR-197GSP2 CATGATCAGCTGGGCCAAGAGCTGGGTGG miR-197RP TT+CA+CCACCTTCTC -0.289 8.2822 0.71 7.10 SEQ ID NO: 263 SEQ ID NO: 264 miR-198 miR-198GSP3 CATGATCAGCTGGGCCAAGACCTATCTC miR-198RP G+GT+CCAGAGGGGAG -0.2986 8.1359 0.31 3.15 SEQ ID NO: 265 SEQ ID NO: 266 miR- miR- CATGATCAGCTGGGCCAAGAAACCAATGT miR- T+AC+AGTAGTCTGCAC -0.3029 9.0509 0.25 2.52 199a* 199a*GSP2 SEQ ID NO: 267 199a*RP SEQ ID NO: 268 miR-199a miR-199aGSP2 CATGATCAGCTGGGCCAAGAGAACAGGTA miR-199aRP C+CC+AGTGTTCAGAC -0.3187 9.2268 0.12 1.16 SEQ ID NO: 269 SEQ ID NO: 270 miR-199b miR-199bGSP CATGATCAGCTGGGCCAAGAGAACAGATAG miR-199bRP C+CC+AGTGTTTAGAC -0.3165 9.3935 2.00 20.04 SEQ ID NO: 271 SEQ ID NO: 272 miR-200a miR-200aGSP2 CATGATCAGCTGGGCCAAGAACATCGTTA miR-200aRP TAA+CAC+TGTCTGGT -0.2754 9.1227 0.08 0.78 SEQ ID NO: 273 SEQ ID NO: 274 miR-200b miR-200bGSP2 CATGATCAGCTGGGCCAAGAGTCATCATT miR-200bRP TAATA+CTG+CCTGGTAAT -0.2935 8.5461 0.08 0.85 SEQ ID NO: 275 SEQ ID NO: 276 miR-202 miR-202 CATGATCAGCTGGGCCAAGATTTTCCCATG miR-202RP# A+GA+GGTATA+GGGCAT -0.2684 9.056 0.25 2.48 GSP10# SEQ ID NO: 277 SEQ ID NO: 278 miR-203 miR-203GSP2 CATGATCAGCTGGGCCAAGACTAGTGGTC miR-203RP G+TG+AAATGTTTAGGACC -0.2852 8.1279 1.60 16.03 SEQ ID NO: 279 SEQ ID NO: 280 miR-204 miR-204GSP2 CATGATCAGCTGGGCCAAGAAGGCATAGG miR-204RP T+TC+CCTTTGTCATCC -0.2925 8.7648 0.16 1.59 SEQ ID NO: 281 SEQ ID NO: 282 miR-205 miR-205GSP CATGATCAGCTGGGCCAAGACAGACTCCGG miR-205RP T+CCTT+CATTCCACC -0.304 8.2407 9.21 92.15 SEQ ID NO: 283 SEQ ID NO: 284 miR-206 mir206GSP7# CATGATCAGCTGGGCCAAGACCACACA miR-206RP# T+G+GAA+TGTAAGGAAGTGT -0.2815 8.2206 0.29 2.86 SEQ ID NO: 285 SEQ ID NO: 286 miR-208 miR- CATGATCAGCTGGGCCAAGAACAAGCTTTTTGC miR- ATAA+GA+CG+AGCAAAAAG -0.2072 7.9097 57.75 577.52 208_GSP13# SEQ ID NO: 287 208_RP4# SEQ ID NO: 288 miR-210 miR-210GSP CATGATCAGCTGGGCCAAGATCAGCCGCTG miR-210RP C+TG+TGCGTGTGACA -0.2717 8.249 0.18 1.77 SEQ ID NO: 289 SEQ ID NO: 290 miR-211 miR-211GSP2 CATGATCAGCTGGGCCAAGAAGGCGAAGG miR-211RP T+TC+CCTTTGTCATCC -0.2926 8.3106 0.10 1.00 SEQ ID NO: 291 SEQ ID NO: 292
miR-212 miR-212GSP9# CATGATCAGCTGGGCCAAGAGGCCGTGAC miR- T+AA+CAGTCTCCAGTCA -0.2916 8.0745 0.59 5.86 SEQ ID NO: 293 212RP2# SEQ ID NO: 294 miR-213 miR-213GSP CATGATCAGCTGGGCCAAGAGGTACAATCA miR-213RP A+CC+ATCGACCGTTG -0.2934 8.1848 2.96 29.59 SEQ ID NO: 295 SEQ ID NO: 296 miR-214 miR-214GSP CATGATCAGCTGGGCCAAGACTGCCTGTCT miR-214RP A+CA+GCAGGCACAGA -0.2947 7.82 0.84 8.44 SEQ ID NO: 297 SEQ ID NO: 298 miR-215 miR-215GSP2 CATGATCAGCTGGGCCAAGAGTCTGTCAA miR-215RP A+TGA+CCTATGAATTGAC -0.2932 8.9273 1.51 15.05 SEQ ID NO: 299 SEQ ID NO: 300 miR-216 miR-216GSP9# CATGATCAGCTGGGCCAAGACACAGTTGC mir216RP# TAA+TCT+CAGCTGGCA -0.273 8.5829 0.95 9.50 SEQ ID NO: 301 SEQ ID NO: 302 miR-217 miR-217GSP2 CATGATCAGCTGGGCCAAGAATCCAATCA miR-217RP2 T+AC+TGCATCAGGAACTGA -0.3089 9.6502 0.07 0.71 SEQ ID NO: 303 SEQ ID NO: 304 miR-218 miR-218GSP2 CATGATCAGCTGGGCCAAGAACATGGTTA miR-218RP TTG+TGCTT+GATCTAAC -0.2778 8.4363 1.00 10.05 SEQ ID NO: 305 SEQ ID NO: 306 miR-220 miR-220GSP CATGATCAGCTGGGCCAAGAAAAGTGTCAG miR-220RP C+CA+CACCGTATCTG -0.2755 9.0728 8.88 88.75 SEQ ID NO: 307 SEQ ID NO: 308 miR-221 miR-221GSP9# CATGATCAGCTGGGCCAAGAGAAACCCAG miR-221RP# A+GC+TACATTGTCTGC -0.2886 8.5743 0.12 1.17 SEQ ID NO: 309 SEQ ID NO: 310 miR-222 miR-222GSP8# CATGATCAGCTGGGCCAAGAGAGACCCA miR-222RP# A+GC+TACATCTGGCT -0.283 8.91 1.64 16.41 SEQ ID NO: 311 SEQ ID NO: 312 miR-223 miR-223GSP CATGATCAGCTGGGCCAAGAGGGGTATTTG miR-223RP TG+TC+AGTTTGTCAAA -0.2998 8.6669 0.94 9.44 SEQ ID NO: 313 SEQ ID NO: 314 miR-224 miR-224GSP8# CATGATCAGCTGGGCCAAGATAAACGGA miR- C+AAG+TCACTAGTGGTT -0.2802 7.5575 0.56 5.63 SEQ ID NO: 315 224RP2# SEQ ID NO: 316 miR-296 miR-296GSP9# CATGATCAGCTGGGCCAAGAACAGGATTG miR- A+GG+GCCCCCCCTCAA -0.3178 8.3856 0.10 0.96 SEQ ID NO: 317 296RP2# SEQ ID NO: 318 miR-299 miR-299GSP9# CATGATCAGCTGGGCCAAGAATGTATGTG miR-299RP# T+GG+TTTACCGTCCC -0.3155 7.9383 1.30 12.96 SEQ ID NO: 319 SEQ ID NO: 320 miR-301 miR-301GSP CATGATCAGCTGGGCCAAGAGCTTTGACAA miR-301RP C+AG+TGCAATAGTATTGT -0.2839 8.314 2.55 25.52 SEQ ID NO: 321 SEQ ID NO: 322 miR- miR-302a*GSP CATGATCAGCTGGGCCAAGAAAAGCAAGTA miR- TAAA+CG+TGGATGTAC -0.2608 8.3921 0.04 0.41 302a* SEQ ID NO: 323 302a*RP SEQ ID NO: 324 miR-302a miR-302aGSP CATGATCAGCTGGGCCAAGATCACCAAAAC miR-302aRP T+AAG+TGCTTCCATGT -0.2577 9.6657 2.17 21.67 SEQ ID NO: 325 SEQ ID NO: 326 miR- miR-302b*GSP CATGATCAGCTGGGCCAAGAAGAAAGCACT miR- A+CTTTAA+CATGGAAGTG -0.2702 8.5153 0.02 0.24 302b* SEQ ID NO: 327 302b*RP SEQ ID NO: 328 miR-302b miR-302bGSP CATGATCAGCTGGGCCAAGACTACTAAAAC miR-302bRP T+AAG+TGCTTCCATGT -0.2398 9.1459 5.11 51.11 SEQ ID NO: 329 SEQ ID NO: 330 miR-302d miR-302dGSP CATGATCAGCTGGGCCAAGAACACTCAAAC miR-302dRP T+AAG+TGCTTCCATGT -0.2368 8.5602 5.98 59.78 SEQ ID NO: 331 SEQ ID NO: 332 miR- miR- CATGATCAGCTGGGCCAAGACAGCAGGTA miR- TT+TAA+CAT+GGGGGTACC -0.312 8.2904 0.33 3.28 302c* 302c*_GSP9# SEQ ID NO: 333 302c*_RP2# SEQ ID NO: 334 miR-302c miR- CATGATCAGCTGGGCCAAGACCACTGAAA miR- T+AAG+TGCTTCCATGTTTCA -0.2945 8.381 14.28 142.76 302cGSP9# SEQ ID NO: 335 302cRP5# SEQ ID NO: 336 miR-320 miR- CATGATCAGCTGGGCCAAGATTCGCCCT miR- AAAA+GCT+GGGTTGAGAGG -0.2677 7.8956 6.73 67.29 320_GSP8# SEQ ID NO: 337 320_RP3# SEQ ID NO: 338 miR-323 miR-323GSP CATGATCAGCTGGGCCAAGAAGAGGTCGAC miR-323RP G+CA+CATTACACGGT -0.2878 8.2546 0.19 1.92 SEQ ID NO: 339 SEQ ID NO: 340 miR-324- miR-324- CATGATCAGCTGGGCCAAGACCAGCAGCAC miR-324- C+CA+CTGCCCCAGGT -0.2698 8.5223 2.54 25.41 3p 3pGSP SEQ ID NO: 341 3pRP SEQ ID NO: 342 miR-324- miR-324- CATGATCAGCTGGGCCAAGAACACCAATGC miR-324- C+GC+ATCCCCTAGGG -0.2861 7.6865 0.06 0.62 5p 5pGSP SEQ ID NO: 343 5pRP SEQ ID NO: 344 miR-325 miR-325GSP CATGATCAGCTGGGCCAAGAACACTTACTG miR-325RP C+CT+AGTAGGTGTCC -0.2976 8.1925 0.01 0.14 SEQ ID NO: 345 SEQ ID NO: 346 miR-326 miR-326GSP CATGATCAGCTGGGCCAAGACTGGAGGAAG miR-326RP C+CT+CTGGGCCCTTC -0.2806 7.897 0.59 5.87 SEQ ID NO: 347 SEQ ID NO: 348 miR-328 miR-328GSP CATGATCAGCTGGGCCAAGAACGGAAGGGC miR-328RP C+TG+GCCCTCTCTGC -0.293 7.929 3.17 31.69 SEQ ID NO: 349 SEQ ID NO: 350 miR-330 miR-330GSP CATGATCAGCTGGGCCAAGATCTCTGCAGG miR-330RP G+CA+AAGCACACGGC -0.3009 7.7999 0.13 1.30 SEQ ID NO: 351 SEQ ID NO: 352 miR-331 miR-331GSP CATGATCAGCTGGGCCAAGATTCTAGGATA miR-331RP G+CC+CCTGGGCCTAT -0.2816 8.1643 0.45 4.54 SEQ ID NO: 353 SEQ ID NO: 354 miR-337 miR-337GSP CATGATCAGCTGGGCCAAGAAAAGGCATCA miR-337RP T+CC+AGCTCCTATATG -0.2968 8.7313 0.10 1.02 SEQ ID NO: 355 SEQ ID NO: 356 miR-338 miR-338GSP CATGATCAGCTGGGCCAAGATCAACAAAAT miR-338RP2 T+CC+AGCATCAGTGATTT -0.2768 8.5618 0.52 5.17 SEQ ID NO: 357 SEQ ID NO: 358 miR-339 miR-339GSP9# CATGATCAGCTGGGCCAAGATGAGCTCCT miR- T+CC+CTGTCCTCCAGG -0.303 8.4873 0.27 2.72 SEQ ID NO: 359 339RP2# SEQ ID NO: 360 miR-340 miR-340GSP CATGATCAGCTGGGCCAAGAGGCTATAAAG miR-340RP TC+CG+TCTCAGTTAC -0.2846 9.6673 0.15 1.45 SEQ ID NO: 361 SEQ ID NO: 362 miR-342 miR-342GSP3 CATGATCAGCTGGGCCAAGAGACGGGTG miR-342RP T+CT+CACACAGAAATCG -0.293 8.1553 4.69 46.85 SEQ ID NO: 363 SEQ ID NO: 364 miR-345 miR-345GSP CATGATCAGCTGGGCCAAGAGCCCTGGACT miR-345RP T+GC+TGACTCCTAGT -0.2909 8.468 0.04 0.40 SEQ ID NO: 365 SEQ ID NO: 366 miR-346 miR-346GSP CATGATCAGCTGGGCCAAGAAGAGGCAGGC miR-346RP T+GT+CTGCCCGCATG -0.2959 8.1958 0.25 2.54 SEQ ID NO: 367 SEQ ID NO: 368 miR-363 miR-363 CATGATCAGCTGGGCCAAGATACAGATGGA miR-363RP# AAT+TG+CAC+GGTATCC -0.2362 8.9762 0.44 4.36 GSP10# SEQ ID NO: 369 SEQ ID NO: 370 miR-367 miR-367GSP CATGATCAGCTGGGCCAAGATCACCATTGC miR-367RP AAT+TG+CACTTTAGCAAT -0.2819 8.6711 0.00 0.03 SEQ ID NO: 371 SEQ ID NO: 372 miR-368 miR-368GSP CATGATCAGCTGGGCCAAGAAAACGTGGAA miR-368RP2 A+CATAGA+GGAAATTCCAC -0.2953 8.0067 6.01 60.11 SEQ ID NO: 373 SEQ ID NO: 374 miR-370 miR-370GSP CATGATCAGCTGGGCCAAGACCAGGTTCCA miR-370RP G+CC+TGCTGGGGTGG -0.2825 8.3162 1.45 14.55 SEQ ID NO: 375 SEQ ID NO: 376 miR-371 miR-371GSP CATGATCAGCTGGGCCAAGAACACTCAAAA miR-371RP G+TG+CCGCCATCTTT -0.295 7.8812 2.51 25.12 SEQ ID NO: 377 SEQ ID NO: 378 miR-372 miR-372GSP CATGATCAGCTGGGCCAAGAACGCTCAAAT miR-372RP A+AA+GTGCTGCGACA -0.2984 8.9183 0.05 0.53 SEQ ID NO: 379 SEQ ID NO: 380 miR-373* miR-373*GSP CATGATCAGCTGGGCCAAGAGGAAAGCGCC miR-373*RP A+CT+CAAAATGGGGG -0.2705 8.4513 0.20 1.99 SEQ ID NO: 381 SEQ ID NO: 382 miR-373 miR-373GSP CATGATCAGCTGGGCCAAGAACACCCCAAA miR-373RP2 GA+AG+TGCTTCGATTTTGG -0.307 7.9056 9.13 91.32 SEQ ID NO: 383 SEQ ID NO: 384 miR-374 miR-374GSP2 CATGATCAGCTGGGCCAAGACACTTATCA miR-374RP TT+AT+AATA+CAACCTGATA -0.2655 9.3795 9.16 91.60 SEQ ID NO: 385 AG SEQ ID NO: 386 miR-375 miR-375GSP CATGATCAGCTGGGCCAAGATCACGCGAGC miR-375RP TT+TG+TTCGTTCGGC -0.3041 8.1181 0.09 0.90 SEQ ID NO: 387 SEQ ID NO: 388 miR-376b miR-376b CATGATCAGCTGGGCCAAGAAACATGGA miR- AT+CAT+AGA+GGAAAATCCA -0.2934 9.0188 1.07 10.74 GSP8# SEQ ID NO: 389 376bRP# SEQ ID NO: 390 miR-378 miR-378GSP CATGATCAGCTGGGCCAAGAACACAGGACC miR-378RP C+TC+CTGACTCCAGG -0.2899 8.1467 0.07 0.73 SEQ ID NO: 391 SEQ ID NO: 392 miR-379 miR- CATGATCAGCTGGGCCAAGATACGTTC miR- T+GGT+AGACTATGGAACG -0.2902 8.2149 10.89 108.86 379_GSP7# SEQ ID NO: 393 379RP2# SEQ ID NO: 394 miR-380- miR-380- CATGATCAGCTGGGCCAAGAGCGCATGTTC miR-380- T+GGT+TGACCATAGA -0.2462 9.4324 1.30 13.04 5p 5pGSP SEQ ID NO: 395 5pRP SEQ ID NO: 396 miR-380- miR-380- CATGATCAGCTGGGCCAAGAAAGATGTGGA miR-380- TA+TG+TAATATGGTCCACA -0.3037 8.0356 3.69 36.89 3p 3pGSP SEQ ID NO: 397 3pRP SEQ ID NO: 398 miR-381 miR-381GSP2 CATGATCAGCTGGGCCAAGAACAGAGAGC miR-381RP2 TATA+CAA+GGGCAAGCT -0.3064 8.8704 1.72 17.16 SEQ ID NO: 399 SEQ ID NO: 400 miR-382 miR-382GSP CATGATCAGCTGGGCCAAGACGAATCCACC miR-382RP G+AA+GTTGTTCGTGGT -0.2803 7.6738 0.66 6.57 SEQ ID NO: 401 SEQ ID NO: 402 miR-383 miR-383GSP CATGATCAGCTGGGCCAAGAAGCCACAATC miR-383RP2 A+GATC+AGAAGGTGATTGT -0.2866 8.1463 0.54 5.45 SEQ ID NO: 403 SEQ ID NO: 404 miR-410 miR-410 CATGATCAGCTGGGCCAAGAACAGGCCAT miR-410RP# AA+TA+TAA+CA+CAGATGGC -0.2297 8.5166 4.27 42.71 GSP9# SEQ ID NO: 405 SEQ ID NO: 406 miR-412 miR-412 CATGATCAGCTGGGCCAAGAACGGCTAGTG miR-412RP# A+CTT+CACCTGGTCCACTA -0.3001 7.9099 4.24 42.37 GSP10# SEQ ID NO: 407 SEQ ID NO: 408 miR-422a miR-422aGSP CATGATCAGCTGGGCCAAGAGGCCTTCTGA miR-422aRP C+TG+GACTTAGGGTC -0.3079 9.3108 5.95 59.54 SEQ ID NO: 409 SEQ ID NO: 410 miR-422b miR-422bGSP CATGATCAGCTGGGCCAAGAGGCCTTCTGA miR-422bRP C+TG+GACTTGGAGTC -0.2993 8.9437 4.86 48.56 SEQ ID NO: 411 SEQ ID NO: 412 miR-423 miR-423GSP CATGATCAGCTGGGCCAAGACTGAGGGGCC miR-423RP A+GC+TCGGTCTGAGG -0.3408 9.2274 6.06 60.62 SEQ ID NO: 413 SEQ ID NO: 414 miR-424 miR-424GSP# CATGATCAGCTGGGCCAAGATTCAAAACAT miR- C+AG+CAGCAATTCATGTTTT -0.3569 9.3419 10.78 107.85 SEQ ID NO: 415 424RP2# SEQ ID NO: 416 miR-425 miR-425GSP CATGATCAGCTGGGCCAAGAGGCGGACACG miR-425RP
A+TC+GGGAATGTCGT -0.2932 7.9786 0.39 3.93 SEQ ID NO: 417 SEQ ID NO: 418 miR-429 miR- CATGATCAGCTGGGCCAAGAACGGTTTTACC miR- T+AATAC+TG+TCTGGTAAAA -0.2458 8.2805 16.21 162.12 429_GSP11# SEQ ID NO: 419 429RP5# SEQ ID NO: 420 miR-431 miR-431 CATGATCAGCTGGGCCAAGATGCATGACGG miR-431RP# T+GT+CTTGCAGGCCG -0.3107 7.7127 7.00 70.05 GSP10# SEQ ID NO: 421 SEQ ID NO: 422 miR-448 miR-448GSP CATGATCAGCTGGGCCAAGAATGGGACATC miR-448RP TTG+CATA+TGTAGGATG -0.3001 8.4969 0.12 1.16 SEQ ID NO: 423 SEQ ID NO: 424 miR-449 miR- CATGATCAGCTGGGCCAAGAACCAGCTAAC miR- T+GG+CAGTGTATTGTTAGC -0.3225 8.4953 2.57 25.70 449GSP10# SEQ ID NO: 425 449RP2# SEQ ID NO: 426 miR-450 miR-450GSP CATGATCAGCTGGGCCAAGATATTAGGAAC miR-450RP TTTT+TG+CGATGTGTT -0.2906 8.1404 0.48 4.82 SEQ ID NO: 427 SEQ ID NO: 428 miR-451 miR-451 CATGATCAGCTGGGCCAAGAAAACTCAGTA miR-451RP# AAA+CCG+TTA+CCATTACTGA -0.2544 8.0291 1.73 17.35 GSP10# SEQ ID NO: 429 SEQ ID NO: 430 let7a let7a-GSP2# CATGATCAGCTGGGCCAAGAAACTATAC let7a-RP# T+GA+GGTAGTAGGTTG -0.3089 9.458 0.04 0.38 SEQ ID NO: 431 SEQ ID NO: 432 let7b let7b-GSP2# CATGATCAGCTGGGCCAAGAAACCACAC let7b-RP# T+GA+GGTAGTAGGTTG -0.2978 7.9144 0.05 0.54 SEQ ID NO: 433 SEQ ID NO: 432 let7c let7c-GSP2# CATGATCAGCTGGGCCAAGAAACCATAC let7c-RP# T+GA+GGTAGTAGGTTG -0.308 7.9854 0.01 0.14 SEQ ID NO: 434 SEQ ID NO: 432 let7d let7d-GSP2# CATGATCAGCTGGGCCAAGAACTATGCA let7d-RP# A+GA+GGTAGTAGGTTG -0.3238 8.3359 0.06 0.57 SEQ ID NO: 435 SEQ ID NO: 436 let7e let7e-GSP2# CATGATCAGCTGGGCCAAGAACTATACA let7e-RP# T+GA+GGTAGGAGGTTG -0.3284 9.7594 0.22 2.20 SEQ ID NO: 437 SEQ ID NO: 438 let7f let7f-GSP2# CATGATCAGCTGGGCCAAGAAACTATAC let7f-RP# T+GA+GGTAGTAGATTG -0.2901 11.107 0.32 3.18 SEQ ID NO: 439 SEQ ID NO: 440 let7g let7g-GSP2# CATGATCAGCTGGGCCAAGAACTGTACA let7g-RP# T+GA+GGTAGTAGTTTG -0.3469 9.8235 0.16 1.64 SEQ ID NO: 441 SEQ ID NO: 442 let7i let7i-GSP2# CATGATCAGCTGGGCCAAGAACAGCACA let7i-RP# T+GA+GGTAGTAGTTTG -0.321 10.82 0.20 1.99 SEQ ID NO: 443 SEQ ID NO: 444 miR-377 miR-377GSP CATGATCAGCTGGGCCAAGAACAAAAGTTG miR-377RP2 AT+CA+CACAAAGGCAAC -0.2979 10.612 13.45 134.48 SEQ ID NO: 445 SEQ ID NO: 446 miR-376a miR- CATGATCAGCTGGGCCAAGAACGTGGA miR- AT+CAT+AGA+GGAAAATCC -0.2938 10.045 63.00 630.00 376a_GSP7 SEQ ID NO: 447 376a_RP5 SEQ ID NO: 448 miR-22 miR-22GSP CATGATCAGCTGGGCCAAGAACAGTTCTTC miR-22RP A+AG+CTGCCAGTTGA -0.2862 8.883 20.46 204.58 SEQ ID NO: 449 SEQ ID NO: 450 miR-200c miR-200cGSP2 CATGATCAGCTGGGCCAAGACCATCATTA miR-200cRP T+AA+TACTGCCGGGT -0.3094 11.5 15.99 159.91 SEQ ID NO: 451 SEQ ID NO: 452 miR-24 miR-24GSP CATGATCAGCTGGGCCAAGACTGTTCCTGC miR-24RP T+GG+CTCAGTTCAGC -0.3123 8.6824 24.34 243.38 SEQ ID NO: 453 SEQ ID NO: 454 miR- miR-29cGSP10 CATGATCAGCTGGGCCAAGAACCGATTTCA miR-29cRP T+AG+CACCATTTGAAAT -0.2975 8.8441 23.22 232.17 29cDNA SEQ ID NO: 455 SEQ ID NO: 456 miR-18 miR-18GSP CATGATCAGCTGGGCCAAGATATCTGCACT miR-18RP T+AA+GGTGCATCTAGT -0.3209 9.0999 14.90 149.01 SEQ ID NO: 457 SEQ ID NO: 458 miR-185 miR-185GSP CATGATCAGCTGGGCCAAGAGAACTGCCTT miR-185RP T+GG+AGAGAAAGGCA -0.3081 8.9289 15.73 157.32 SEQ ID NO: 459 SEQ ID NO: 460 miR-181b miR- CATGATCAGCTGGGCCAAGACCCACCGA miR- AA+CATT+CATTGCTGTC -0.3115 10.846 15.87 158.67 181bGSP8# SEQ ID NO: 461 181bRP2# SEQ ID NO: 462 miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR- TCACAGTGAACCGGT approx. approx. approx. approx. SEQ ID NO: 161 128anLRP SEQ ID NO: 494 -0.2866 8.0867 0.16 1.60 miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR- AGCTGGTGTTGTGAA approx. approx. approx. approx. SEQ ID NO: 187 138nLRP SEQ ID NO: 495 -0.3023 9.0814 0.22 2.19 miR-143 miR-143GSP8# CATGATCAGCTGGGCCAAGATGAGCTAC miR- TGAGATGAAGCACTGT approx. approx. approx. approx. SEQ ID NO: 197 143nLRP SEQ ID NO: 496 -0.3008 9.2675 0.37 3.71 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR- TCTCCCAACCCTTGTA approx. approx. approx. approx. SEQ ID NO: 213 150nLRP SEQ ID NO: 497 -0.2943 8.3945 0.06 0.56 miR-181a miR- CATGATCAGCTGGGCCAAGAACTCACCGA miR- AACATTCAACGCTGT approx. approx. approx. approx. 181aGSP9# SEQ ID NO: 227 181anLRP SEQ ID NO: 498 -0.2919 7.968 1.70 17.05 miR-194 mir194GSP8# CATGATCAGCTGGGCCAAGATCCACATG miR- TGTAACAGCAACTCCA approx. approx. approx. approx. SEQ ID NO: 255 194nLRP SEQ ID NO: 499 -0.3078 8.8045 0.37 3.69 #denotes primers for assays that required extensive testing and primer design modification to achieve optimal assay results including high sensitivity and high dynamic range.
Example 4
[0154] This Example describes assays and primers designed for quantitative analysis of murine miRNA expression patterns.
[0155] Methods: The representative murine microRNA target templates described in TABLE 7 are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the "miRBase sequence database" as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34: D140-D144. As indicated below in TABLE 7, the murine microRNA templates are either totally identical to the corresponding human microRNA templates, identical in the overlapping sequence with differing ends, or contain one or more base pair changes as compared to the human microRNA sequence. The murine microRNA templates that are identical or that have identical overlapping sequence to the corresponding human templates can be assayed using the same primer sets designed for the human microRNA templates, as indicated in TABLE 7. For the murine microRNA templates with one or more base pair changes in comparison to the corresponding human templates, primer sets have been designed specifically for detection of the murine microRNA, and these primers are provided in TABLE 7. The extension primer reaction and quantitative PCR reactions for detection of the murine microRNA templates may be carried out as described in EXAMPLE 3.
TABLE-US-00009 TABLE 7 PRIMERS TO DETECT MURINE MICRORNA TARGET TEMPLATES Mouse Target Mouse microRNA as compared microRNA: Extension Primer Name Extension Primer Sequence Reverse Primer Name Reverse Primer Sequence to Human microRNA miR-1 miR1GSP10 CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP T+G+GAA+TG+TAAAGAAGT Identical SEQ ID NO: 47 SEQ ID NO: 48 miR-7 miR-7GSP10 CATGATCAGCTGGGCCAAGAAACAAAATC miR-7_RP6 T+GGAA+GACTTGTGATTTT one or more base pairs differ SEQ ID NO: 486 SEQ ID NO: 487 miR-9* miR-9*GSP CATGATCAGCTGGGCCAAGAACTTTCGGTT miR-9*RP TAAA+GCT+AGATAACCG Identical overlapping sequence, SEQ ID NO: 51 SEQ ID NO: 52 ends differ miR-10a miR-10aGSP CATGATCAGCTGGGCCAAGACACAAATTCG miR-10aRP T+AC+CCTGTAGATCCG Identical SEQ ID NO: 53 SEQ ID NO: 54 miR-10b miR-10b_GSP11 CATGATCAGCTGGGCCAAGAACACAAATTCG miR-10b_RP2 C+CC+TGT+AGAACCGAAT one or more base pairs differ SEQ ID NO: 492 SEQ ID NO: 493 miR-15a miR-15aGSP CATGATCAGCTGGGCCAAGACACAAACCAT miR-15aRP T+AG+CAGCACATAATG Identical SEQ ID NO: 57 SEQ ID NO: 58 miR-15b miR-15bGSP2 CATGATCAGCTGGGCCAAGATGTAAACCA miR-15bRP T+AG+CAGCACATCAT Identical SEQ ID NO: 59 SEQ ID NO: 60 miR-16 miR-16GSP2 CATGATCAGCTGGGCCAAGACGCCAATAT miR-16RP T+AG+CAGCACGTAAA Identical SEQ ID NO: 61 SEQ ID NO: 62 miR-17-3p miR-17-3pGSP CATGATCAGCTGGGCCAAGAACAAGTGCCC miR-17-3pRP A+CT+GCAGTGAGGGC one or more base pairs differ SEQ ID NO: 463 SEQ ID NO: 464 miR-17-5p miR-17-5pGSP2 CATGATCAGCTGGGCCAAGAACTACCTGC miR-17-5pRP C+AA+AGTGCTTACAGTG Identical SEQ ID NO: 65 SEQ ID NO: 66 miR-19a miR-19aGSP2 CATGATCAGCTGGGCCAAGATCAGTTTTG miR-19aRP TG+TG+CAAATCTATGC Identical SEQ ID NO: 67 SEQ ID NO: 68 miR-19b miR-19bGSP CATGATCAGCTGGGCCAAGATCAGTTTTGC miR-19bRP TG+TG+CAAATCCATG Identical SEQ ID NO: 69 SEQ ID NO: 70 miR-20 miR-20GSP3 CATGATCAGCTGGGCCAAGACTACCTGC miR-20RP T+AA+AGTGCTTATAGTGCA Identical SEQ ID NO: 71 SEQ ID NO: 72 miR-21 miR-21GSP2 CATGATCAGCTGGGCCAAGATCAACATCA miR-21RP T+AG+CTTATCAGACTGATG Identical SEQ ID NO: 73 SEQ ID NO: 74 miR-23a miR-23aGSP CATGATCAGCTGGGCCAAGAGGAAATCCCT miR-23aRP A+TC+ACATTGCCAGG Identical SEQ ID NO: 75 SEQ ID NO: 76 miR-23b miR-23bGSP CATGATCAGCTGGGCCAAGAGGTAATCCCT miR-23bRP A+TC+ACATTGCCAGG Identical SEQ ID NO: 77 SEQ ID NO: 78 miR-24 miR-24P5 CATGATCAGCTGGGCCAAGACTGTTCCTGC miR24-1,2R TGG+CTCAGTTCAGC Identical TG SEQ ID NO: 19 SEQ ID NO: 7 miR-25 miR-25GSP CATGATCAGCTGGGCCAAGATCAGACCGAG miR-25RP C+AT+TGCACTTGTCTC Identical SEQ ID NO: 79 SEQ ID NO: 80 miR-26a miR-26aGSP9 CATGATCAGCTGGGCCAAGAGCCTATCCT miR-26aRP2 TT+CA+AGTAATCCAGGAT Identical SEQ ID NO: 81 SEQ ID NO: 82 miR-26b miR-26bGSP9 CATGATCAGCTGGGCCAAGAAACCTATCC miR-26bRP2 TT+CA+AGT+AATTCAGGAT Identical SEQ ID NO: 83 SEQ ID NO: 84 miR-27a miR-27aGSP CATGATCAGCTGGGCCAAGAGCGGAACTTA miR-27aRP TT+CA+CAGTGGCTAA Identical SEQ ID NO: 85 SEQ ID NO: 86 miR-27b miR-27bGSP CATGATCAGCTGGGCCAAGAGCAGAACTTA miR-27bRP TT+CA+CAGTGGCTAA Identical SEQ ID NO: 87 SEQ ID NO: 88 miR-28 miR-28GSP CATGATCAGCTGGGCCAAGACTCAATAGAC miR-28RP A+AG+GAGCTCACAGT Identical SEQ ID NO: 89 SEQ ID NO: 90 miR-29a miR-29aGSP8 CATGATCAGCTGGGCCAAGAAACCGATT miR-29aRP2 T+AG+CACCATCTGAAAT Identical SEQ ID NO: 91 SEQ ID NO: 92 miR-29b miR-29bGSP2 CATGATCAGCTGGGCCAAGAAACACTGAT miR-29bRP2 T+AG+CACCATTTGAAATCAG Identical SEQ ID NO: 93 SEQ ID NO: 94 miR-30a-5p miR-30a-5pGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30a-5pRP T+GT+AAACATCCTCGAC Identical SEQ ID NO: 95 SEQ ID NO: 96 miR-30b miR-30bGSP CATGATCAGCTGGGCCAAGAAGCTGAGTGT miR-30bRP TGT+AAA+CATCCTACACT Identical SEQ ID NO: 97 SEQ ID NO: 98 miR-30c miR-30cGSP CATGATCAGCTGGGCCAAGAGCTGAGAGTG miR-30cRP TGT+AAA+CATCCTACACT Identical SEQ ID NO: 99 SEQ ID NO: 100 miR-30d miR-30dGSP CATGATCAGCTGGGCCAAGACTTCCAGTCG miR-30dRP T+GTAAA+CATCCCCG Identical SEQ ID NO: 101 SEQ ID NO: 102 miR-30e-3p miR-30e-3pGSP9 CATGATCAGCTGGGCCAAGAGCTGTAAAC miR-30e-3pRP5 CTTT+CAGT+CGGATGTTT Identical SEQ ID NO: 103 SEQ ID NO: 104 miR-31 miR-31GSP CATGATCAGCTGGGCCAAGACAGCTATGCC miR-31RP G+GC+AAGATGCTGGC Identical overlapping sequence, SEQ ID NO: 107 SEQ ID NO: 108 ends differ miR-32 miR-32GSP CATGATCAGCTGGGCCAAGAGCAACTTAGT miR-32RP TATTG+CA+CATTACTAAG Identical SEQ ID NO: 109 SEQ ID NO: 110 miR-33 miR-33GSP2 CATGATCAGCTGGGCCAAGACAATGCAAC miR-33RP G+TG+CATTGTAGTTGC Identical SEQ ID NO: 111 SEQ ID NO: 112 miR-34a miR-34aGSP CATGATCAGCTGGGCCAAGAAACAACCAGC miR-34aRP T+GG+CAGTGTCTTAG Identical SEQ ID NO: 113 SEQ ID NO: 114 miR-34b miR-34bGSP CATGATCAGCTGGGCCAAGACAATCAGCTA miR-34bRP TA+GG+CAGTGTAATT one or more base pairs differ SEQ ID NO: 115 SEQ ID NO: 482 miR-34c miR-34cGSP CATGATCAGCTGGGCCAAGAGCAATCAGCT miR-34cRP A+GG+CAGTGTAGTTA Identical SEQ ID NO: 117 SEQ ID NO: 118 miR-92 miR-92GSP CATGATCAGCTGGGCCAAGACAGGCCGGGA miR-92RP T+AT+TGCACTTGTCCC Identical SEQ ID NO: 119 SEQ ID NO: 120 miR-93 miR-93GSP CATGATCAGCTGGGCCAAGACTACCTGCAC miR-93RP AA+AG+TGCTGTTCGT Identical overlapping sequence, SEQ ID NO: 121 SEQ ID NO: 122 ends differ miR-96 miR-96GSP CATGATCAGCTGGGCCAAGAGCAAAAATGT miR-96RP T+TT+GGCACTAGCAC Identical overlapping sequence, SEQ ID NO: 125 SEQ ID NO: 126 ends differ miR-98 miR-98GSP CATGATCAGCTGGGCCAAGAAACAATACAA miR-98RP TGA+GGT+AGTAAGTTG Identical SEQ ID NO: 127 SEQ ID NO: 128 miR-99a miR-99aGSP CATGATCAGCTGGGCCAAGACACAAGATCG miR-99aRP A+AC+CCGTAGATCCG Identical overlapping sequence, SEQ ID NO: 129 SEQ ID NO: 130 ends differ miR-99b miR-99bGSP CATGATCAGCTGGGCCAAGACGCAAGGTCG miR-99bRP C+AC+CCGTAGAACCG Identical SEQ ID NO: 131 SEQ ID NO: 132 miR-100 miR-100GSP CATGATCAGCTGGGCCAAGACACAAGTTCG miR-100RP A+AC+CCGTAGATCCG Identical SEQ ID NO: 133 SEQ ID NO: 134 miR-101 miR-101GSP CATGATCAGCTGGGCCAAGACTTCAGTTAT miR-101RP TA+CAG+TACTGTGATAACT Identical SEQ ID NO: 135 SEQ ID NO: 136 miR-103 miR-103GSP CATGATCAGCTGGGCCAAGATCATAGCCCT miR-103RP A+GC+AGCATTGTACA Identical SEQ ID NO: 137 SEQ ID NO: 138 miR-106a miR-106aGSP CATGATCAGCTGGGCCAAGATACCTGCAC miR-106aRP CAA+AG+TGCTAACAGTG one or more base pairs differ SEQ ID NO: 472 SEQ ID NO: 473 miR-106b miR-106bGSP CATGATCAGCTGGGCCAAGAATCTGCACTG miR-106bRP T+AAAG+TGCTGACAGT Identical SEQ ID NO: 143 SEQ ID NO: 144 miR-107 miR-107GSP8 CATGATCAGCTGGGCCAAGATGATAGCC miR-107RP2 A+GC+AGCATTGTACAG Identical SEQ ID NO: 145 SEQ ID NO: 146 miR-122a miR-122aGSP CATGATCAGCTGGGCCAAGAACAAACACCA miR-122aRP T+GG+AGTGTGACAAT Identical SEQ ID NO: 147 SEQ ID NO: 148 miR-124a miR-124aGSP CATGATCAGCTGGGCCAAGATGGCATTCAC miR-124aRP T+TA+AGGCACGCGGT Identical overlapping sequence, SEQ ID NO: 149 SEQ ID NO: 150 ends differ miR-125a miR-125aGSP CATGATCAGCTGGGCCAAGACACAGGTTAA miR-125aRP T+CC+CTGAGACCCTT Identical SEQ ID NO: 151 SEQ ID NO: 152 miR-125b miR-125bGSP CATGATCAGCTGGGCCAAGATCACAAGTTA miR-125bRP T+CC+CTGAGACCCTA Identical SEQ ID NO: 153 SEQ ID NO: 154 miR-126 miR-126GSP CATGATCAGCTGGGCCAAGAGCATTATTAC miR-126RP T+CG+TACCGTGAGTA Identical SEQ ID NO: 155 SEQ ID NO: 156 miR-126* miR-126*GSP3 CATGATCAGCTGGGCCAAGACGCGTACC miR-126*RP C+ATT+ATTA+CTTTTGGTACG Identical SEQ ID NO: 157 SEQ ID NO: 158 miR-127 miR-127GSP CATGATCAGCTGGGCCAAGAAGCCAAGCTC miR-127RP T+CG+GATCCGTCTGA Identical overlapping sequence, SEQ ID NO: 159 SEQ ID NO: 160 ends differ miR-128a miR-128aGSP CATGATCAGCTGGGCCAAGAAAAAGAGACC miR-128aRP T+CA+CAGTGAACCGG Identical SEQ ID NO: 161 SEQ ID NO: 162 miR-128b miR-128bGSP CATGATCAGCTGGGCCAAGAGAAAGAGACC miR-128bRP T+CA+CAGTGAACCGG Identical SEQ ID NO: 163 SEQ ID NO: 164 miR-130a miR-130aGSP CATGATCAGCTGGGCCAAGAATGCCCTTTT miR-130aRP C+AG+TGCAATGTTAAAAG Identical SEQ ID NO: 167 SEQ ID NO: 168 miR-130b miR-130bGSP CATGATCAGCTGGGCCAAGAATGCCCTTTC miR-130bRP C+AG+TGCAATGATGA Identical SEQ ID NO: 169 SEQ ID NO: 170 miR-132 miR-132GSP CATGATCAGCTGGGCCAAGACGACCATGGC miR-132RP T+AA+CAGTCTACAGCC Identical SEQ ID NO: 171 SEQ ID NO: 172 miR-133a miR-133aGSP CATGATCAGCTGGGCCAAGAACAGCTGGTT miR-133aRP T+TG+GTCCCCTTCAA Identical
SEQ ID NO: 173 SEQ ID NO: 174 miR-133b miR-133bGSP CATGATCAGCTGGGCCAAGATAGCTGGTTG miR-133bRP T+TG+GTCCCCTTCAA Identical SEQ ID NO: 175 SEQ ID NO: 176 miR-134 miR-134GSP CATGATCAGCTGGGCCAAGACCCTCTGGTC miR-134RP T+GT+GACTGGTTGAC Identical overlapping sequence, SEQ ID NO: 177 SEQ ID NO: 178 ends differ miR-135a miR-135aGSP CATGATCAGCTGGGCCAAGATCACATAGGA miR-135aRP T+AT+GGCTTTTTATTCCT Identical SEQ ID NO: 179 SEQ ID NO: 180 miR-135b miR-135bGSP CATGATCAGCTGGGCCAAGACACATAGGAA miR-135bRP T+AT+GGCTTTTCATTCC Identical SEQ ID NO: 181 SEQ ID NO: 182 miR-136 miR-136GSP CATGATCAGCTGGGCCAAGATCCATCATCA miR-136RP A+CT+CCATTTGTTTTGATG Identical SEQ ID NO: 183 SEQ ID NO: 184 miR-137 miR-137GSP CATGATCAGCTGGGCCAAGACTACGCGTAT miR-137RP T+AT+TGCTTAAGAATACGC Identical overlapping sequence, SEQ ID NO: 185 SEQ ID NO: 186 ends differ miR-138 miR-138GSP2 CATGATCAGCTGGGCCAAGACGGCCTGAT miR-138RP A+GC+TGGTGTTGTGA Identical SEQ ID NO: 187 SEQ ID NO: 188 miR-139 miR-139GSP CATGATCAGCTGGGCCAAGAAGACACGTGC miR-139RP T+CT+ACAGTGCACGT Identical SEQ ID NO: 189 SEQ ID NO: 190 miR-140 miR-140GSP CATGATCAGCTGGGCCAAGACTACCATAGG miR-140RP A+GT+GGTTTTACCCT Identical overlapping sequence, SEQ ID NO: 191 SEQ ID NO: 192 ends differ miR-141 miR-141GSP9 CATGATCAGCTGGGCCAAGACCATCTTTA miR-141RP2 TAA+CAC+TGTCTGGTAA Identical SEQ ID NO: 193 SEQ ID NO: 194 miR-142-3p miR-142-3pGSP3 CATGATCAGCTGGGCCAAGATCCATAAA miR-142-3pRP TGT+AG+TGTTTCCTACT Identical overlapping sequence, SEQ ID NO: 195 SEQ ID NO: 196 ends differ miR-143 miR-143GSP8 CATGATCAGCTGGGCCAAGATGAGCTAC miR-143RP2 T+GA+GATGAAGCACTG Identical SEQ ID NO: 197 SEQ ID NO: 198 miR-144 miR-144GSP2 CATGATCAGCTGGGCCAAGACTAGTACAT miR-144RP TA+CA+GTAT+AGATGATG Identical SEQ ID NO: 199 SEQ ID NO: 200 miR-145 miR-145GSP2 CATGATCAGCTGGGCCAAGAAAGGGATTC miR-145RP G+TC+CAGTTTTCCCA Identical SEQ ID NO: 201 SEQ ID NO: 202 miR-146 miR-146GSP3 CATGATCAGCTGGGCCAAGAAACCCATG miR-146RP T+GA+GAACTGAATTCCA Identical SEQ ID NO: 203 SEQ ID NO: 204 miR-148a miR-148aGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148aRP2 T+CA+GTGCACTACAGAACT Identical SEQ ID NO: 207 SEQ ID NO: 208 miR-148b miR-148bGSP2 CATGATCAGCTGGGCCAAGAACAAAGTTC miR-148bRP T+CA+GTGCATCACAG Identical SEQ ID NO: 209 SEQ ID NO: 210 miR-149 miR-149GSP2 CATGATCAGCTGGGCCAAGAGGAGTGAAG miR-149RP T+CT+GGCTCCGTGTC Identical SEQ ID NO: 211 SEQ ID NO: 212 miR-150 miR-150GSP3 CATGATCAGCTGGGCCAAGACACTGGTA miR-150RP T+CT+CCCAACCCTTG Identical SEQ ID NO: 213 SEQ ID NO: 214 miR-151 miR-151GSP2 CATGATCAGCTGGGCCAAGACCTCAAGGA miR-151RP A+CT+AGACTGAGGCTC one or more base pairs differ SEQ ID NO: 215 SEQ ID NO: 477 miR-152 miR-152GSP2 CATGATCAGCTGGGCCAAGACCCAAGTTC miR-152RP T+CA+GTGCATGACAG Identical SEQ ID NO: 217 SEQ ID NO: 218 miR-153 miR-153GSP2 CATGATCAGCTGGGCCAAGATCACTTTTG miR-153RP TTG+CAT+AGTCACAAAA Identical overlapping sequence, SEQ ID NO: 219 SEQ ID NO: 220 ends differ miR-154 miR-154GSP9 CATGATCAGCTGGGCCAAGACGAAGGCAA miR-154RP3 TA+GGTTA+TCCGTGTT Identical SEQ ID NO: 223 SEQ ID NO: 224 miR-155 miR-155GSP8 CATGATCAGCTGGGCCAAGACCCCTATC miR-155RP2 TT+AA+TGCTAATTGTGATAGG one or more base pairs differ SEQ ID NO: 225 SEQ ID NO: 489 miR-181a miR-181aGSP9 CATGATCAGCTGGGCCAAGAACTCACCGA miR-181aRP2 AA+CATT+CAACGCTGTC Identical SEQ ID NO: 227 SEQ ID NO: 228 miR-181c miR-181cGSP9 CATGATCAGCTGGGCCAAGAACTCACCGA miR-181cRP2 AA+CATT+CAACCTGTCG Identical SEQ ID NO: 229 SEQ ID NO: 230 miR-182 miR-182*GSP CATGATCAGCTGGGCCAAGATAGTTGGCAA miR-182*RP T+GG+TTCTAGACTTGC Identical SEQ ID NO: 231 SEQ ID NO: 232 miR-183 miR-183GSP2 CATGATCAGCTGGGCCAAGACAGTGAATT miR-183RP T+AT+GGCACTGGTAG Identical SEQ ID NO: 235 SEQ ID NO: 236 miR-184 miR-184GSP2 CATGATCAGCTGGGCCAAGAACCCTTATC miR-184RP T+GG+ACGGAGAACTG Identical SEQ ID NO: 237 SEQ ID NO: 238 miR-186 miR-186GSP9 CATGATCAGCTGGGCCAAGAAAGCCCAAA miR-186RP3 CA+AA+GAATT+CTCCTTTTGG Identical SEQ ID NO: 239 SEQ ID NO: 240 miR-187 miR-187GSP CATGATCAGCTGGGCCAAGACGGCTGCAAC miR-187RP T+CG+TGTCTTGTGTT Identical overlapping sequence, SEQ ID NO: 241 SEQ ID NO: 242 ends differ miR-188 miR-188GSP CATGATCAGCTGGGCCAAGAACCCTCCACC miR-188RP C+AT+CCCTTGCATGG Identical SEQ ID NO: 243 SEQ ID NO: 244 miR-189 miR-189GSP2 CATGATCAGCTGGGCCAAGAACTGATATC miR-189RP G+TG+CCTACTGAGCT Identical SEQ ID NO: 245 SEQ ID NO: 246 miR-190 miR-190GSP9 CATGATCAGCTGGGCCAAGAACCTAATAT miR-190RP4 T+GA+TA+TGTTTGATATATTAG Identical SEQ ID NO: 247 SEQ ID NO: 248 miR-191 miR-191GSP2 CATGATCAGCTGGGCCAAGAAGCTGCTTT miR-191RP2 C+AA+CGGAATCCCAAAAG Identical SEQ ID NO: 249 SEQ ID NO: 250 miR-192 miR-192GSP2 CATGATCAGCTGGGCCAAGAGGCTGTCAA miR-192RP C+TGA+CCTATGAATTGAC Identical overlapping sequence, SEQ ID NO: 251 SEQ ID NO: 252 ends differ miR-193 miR-193GSP9 CATGATCAGCTGGGCCAAGACTGGGACTT miR-193RP2 AA+CT+GGCCTACAAAG Identical SEQ ID NO: 253 SEQ ID NO: 254 miR-194 mir194GSP8 CATGATCAGCTGGGCCAAGATCCACATG mir194RP TG+TAA+CAGCAACTCCA Identical SEQ ID NO: 255 SEQ ID NO: 256 miR-195 miR-195GSP9 CATGATCAGCTGGGCCAAGAGCCAATATT miR-195RP3 T+AG+CAG+CACAGAAATA Identical SEQ ID NO: 257 SEQ ID NO: 258 miR-196a miR-196aGSP CATGATCAGCTGGGCCAAGACCAACAACAT miR-196aRP TA+GG+TAGTTTCATGTTG Identical SEQ ID NO: 261 SEQ ID NO: 262 miR-196b miR-196bGSP CATGATCAGCTGGGCCAAGACCAACAACAG miR-196bRP TA+GGT+AGTTTCCTGT Identical SEQ ID NO: 259 SEQ ID NO: 260 miR-199a* miR-199a*GSP2 CATGATCAGCTGGGCCAAGAAACCAATGT miR-199a*RP T+AC+AGTAGTCTGCAC Identical SEQ ID NO: 267 SEQ ID NO: 268 miR-199a miR-199aGSP2 CATGATCAGCTGGGCCAAGAGAACAGGTA miR-199aRP C+CC+AGTGTTCAGAC Identical SEQ ID NO: 269 SEQ ID NO: 270 miR-199b miR-199bGSP CATGATCAGCTGGGCCAAGAGAACAGGTAG miR-199bRP C+CC+AGTGTTTAGAC one or more base pairs differ SEQ ID NO: 475 SEQ ID NO: 272 miR-200a miR-200aGSP2 CATGATCAGCTGGGCCAAGAACATCGTTA miR-200aRP TAA+CAC+TGTCTGGT Identical SEQ ID NO: 273 SEQ ID NO: 274 miR-200b miR-200bGSP2 CATGATCAGCTGGGCCAAGAGTCATCATT miR-200bRP TAATA+CTG+CCTGGTAAT Identical SEQ ID NO: 275 SEQ ID NO: 276 miR-203 miR-203GSP2 CATGATCAGCTGGGCCAAGACTAGTGGTC miR-203RP G+TG+AAATGTTTAGGACC Identical overlapping sequence, SEQ ID NO: 279 SEQ ID NO: 280 ends differ miR-204 miR-204GSP2 CATGATCAGCTGGGCCAAGAAGGCATAGG miR-204RP T+TC+CCTTTGTCATCC Identical overlapping sequence, SEQ ID NO: 281 SEQ ID NO: 282 ends differ miR-205 miR-205GSP CATGATCAGCTGGGCCAAGACAGACTCCGG miR-205RP T+CCTT+CATTCCACC Identical SEQ ID NO: 283 SEQ ID NO: 284 miR-206 mir206GSP7 CATGATCAGCTGGGCCAAGACCACACA miR-206RP T+G+GAA+TGTAAGGAAGTGT Identical SEQ ID NO: 285 SEQ ID NO: 286 miR-208 miR-208_GSP13 CATGATCAGCTGGGCCAAGAACAAGCTTTT miR-208_RP4 ATAA+GA+CG+AGCAAAAAG Identical TGC SEQ ID NO: 288 SEQ ID NO: 287 miR-210 miR-210GSP CATGATCAGCTGGGCCAAGATCAGCCGCTG miR-210RP C+TG+TGCGTGTGACA Identical SEQ ID NO: 289 SEQ ID NO: 290 miR-211 miR-211GSP2 CATGATCAGCTGGGCCAAGAAGGCAAAGG miR-211RP T+TC+CCTTTGTCATCC one or more base pairs differ SEQ ID NO: 491 SEQ ID NO: 292 miR-212 miR-212GSP9 CATGATCAGCTGGGCCAAGAGGCCGTGAC miR-212RP2 T+AA+CAGTCTCCAGTCA Identical SEQ ID NO: 293 SEQ ID NO: 294 miR-213 miR-213GSP CATGATCAGCTGGGCCAAGAGGTACAATCA miR-213RP A+CC+ATCGACCGTTG Identical SEQ ID NO: 295 SEQ ID NO: 296 miR-214 miR-214GSP CATGATCAGCTGGGCCAAGACTGCCTGTCT miR-214RP A+CA+GCAGGCACAGA Identical SEQ ID NO: 297 SEQ ID NO: 298 miR-215 miR-215GSP2 CATGATCAGCTGGGCCAAGAGTCTGTCAA miR-215RP A+TGA+CCTATGATTTGAC one or more base pairs differ SEQ ID NO: 299 SEQ ID NO: 469 miR-216 miR-216GSP9 CATGATCAGCTGGGCCAAGACACAGTTGC mir216RP TAA+TCT+CAGCTGGCA Identical SEQ ID NO: 301 SEQ ID NO: 302 miR-217 miR-217GSP2 CATGATCAGCTGGGCCAAGAATCCAGTCA miR-217RP2 T+AC+TGCATCAGGAACTGA one or more base pairs differ SEQ ID NO: 481 SEQ ID NO: 304 miR-218 miR-218GSP2 CATGATCAGCTGGGCCAAGAACATGGTTA miR-218RP TTG+TGCTT+GATCTAAC Identical SEQ ID NO: 305 SEQ ID NO: 306 miR-221 miR-221GSP9 CATGATCAGCTGGGCCAAGAGAAACCCAG miR-221RP A+GC+TACATTGTCTGC Identical overlapping sequence, SEQ ID NO: 309 SEQ ID NO: 310 ends differ miR-222 miR-222GSP8 CATGATCAGCTGGGCCAAGAGAGACCCA miR-222RP A+GC+TACATCTGGCT Identical SEQ ID NO: 311 SEQ ID NO: 312
miR-223 miR-223GSP CATGATCAGCTGGGCCAAGAGGGGTATTTG miR-223RP TG+TC+AGTTTGTCAAA Identical SEQ ID NO: 313 SEQ ID NO: 314 miR-224 miR-224GSP8 CATGATCAGCTGGGCCAAGATAAACGGA miR-224RP2 C+AAG+TCACTAGTGGTT Identical overlapping sequence, SEQ ID NO: 315 SEQ ID NO: 316 ends differ miR-296 miR-296GSP9 CATGATCAGCTGGGCCAAGAACAGGATTG miR-296RP2 A+GG+GCCCCCCCTCAA Identical SEQ ID NO: 317 SEQ ID NO: 318 miR-299 miR-299GSP9 CATGATCAGCTGGGCCAAGAATGTATGTG miR-299RP T+GG+TTTACCGTCCC Identical SEQ ID NO: 319 SEQ ID NO: 320 miR-301 miR-301GSP CATGATCAGCTGGGCCAAGAGCTTTGACAA miR-301RP C+AG+TGCAATAGTATTGT Identical SEQ ID NO: 321 SEQ ID NO: 322 miR-302a miR-302aGSP CATGATCAGCTGGGCCAAGATCACCAAAAC miR-302aRP T+AAG+TGCTTCCATGT Identical SEQ ID NO: 325 SEQ ID NO: 326 miR-320 miR-320_GSP8 CATGATCAGCTGGGCCAAGATTCGCCCT miR-320_RP3 AAAA+GCT+GGGTTGAGAGG Identical SEQ ID NO: 337 SEQ ID NO: 338 miR-323 miR-323GSP CATGATCAGCTGGGCCAAGAAGAGGTCGAC miR-323RP G+CA+CATTACACGGT Identical SEQ ID NO: 339 SEQ ID NO: 340 miR-324-3p miR-324-3pGSP CATGATCAGCTGGGCCAAGACCAGCAGCAC miR-324-3pRP C+CA+CTGCCCCAGGT Identical SEQ ID NO: 341 SEQ ID NO: 342 miR-324-5p miR-324-5pGSP CATGATCAGCTGGGCCAAGAACACCAATGC miR-324-5pRP C+GC+ATCCCCTAGGG Identical overlapping sequence, SEQ ID NO: 343 SEQ ID NO: 344 ends differ miR-325 miR-325GSP CATGATCAGCTGGGCCAAGAACACTTACTG miR-325RP C+CT+AGTAGGTGCTC one or more base pairs differ SEQ ID NO: 345 SEQ ID NO: 476 miR-326 miR-326GSP CATGATCAGCTGGGCCAAGACTGGAGGAAG miR-326RP C+CT+CTGGGCCCTTC Identical overlapping sequence, SEQ ID NO: 347 SEQ ID NO: 348 ends differ miR-328 miR-328GSP CATGATCAGCTGGGCCAAGAACGGAAGGGC miR-328RP C+TG+GCCCTCTCTGC Identical SEQ ID NO: 349 SEQ ID NO: 350 miR-330 miR-330GSP CATGATCAGCTGGGCCAAGATCTCTGCAGG miR-330RP G+CA+AAGCACAGGGC one or more base pairs differ SEQ ID NO: 351 SEQ ID NO: 478 miR-331 miR-331GSP CATGATCAGCTGGGCCAAGATTCTAGGATA miR-331RP G+CC+CCTGGGCCTAT Identical SEQ ID NO: 353 SEQ ID NO: 354 miR-337 miR-337GSP CATGATCAGCTGGGCCAAGAAAAGGCATCA miR-337RP T+TC+AGCTCCTATATG one or more base pairs differ SEQ ID NO: 355 SEQ ID NO: 490 miR-338 miR-338GSP CATGATCAGCTGGGCCAAGATCAACAAAAT miR-338RP2 T+CC+AGCATCAGTGATTT Identical SEQ ID NO: 357 SEQ ID NO: 358 miR-339 miR-339GSP9 CATGATCAGCTGGGCCAAGATGAGCTCCT miR-339RP2 T+CC+CTGTCCTCCAGG Identical SEQ ID NO: 359 SEQ ID NO: 360 miR-340 miR-340GSP CATGATCAGCTGGGCCAAGAGGCTATAAAG miR-340RP TC+CG+TCTCAGTTAC Identical SEQ ID NO: 361 SEQ ID NO: 362 miR-342 miR-342GSP3 CATGATCAGCTGGGCCAAGAGACGGGTG miR-342RP T+CT+CACACAGAAATCG Identical SEQ ID NO: 363 SEQ ID NO: 364 miR-345 miR-345GSP CATGATCAGCTGGGCCAAGAGCACTGGACT miR-345RP T+GC+TGACCCCTAGT one or more base pairs differ SEQ ID NO: 484 SEQ ID NO: 485 miR-346 miR-346GSP CATGATCAGCTGGGCCAAGAAGAGGCAGGC miR-346RP T+GT+CTGCCCGAGTG one or more base pairs differ SEQ ID NO: 367 SEQ ID NO: 488 miR-363 miR-363 GSP10 CATGATCAGCTGGGCCAAGATACAGATGGA miR-363RP AAT+TG+CAC+GGTATCC Identical SEQ ID NO: 369 SEQ ID NO: 370 miR-370 miR-370GSP CATGATCAGCTGGGCCAAGACCAGGTTCCA miR-370RP G+CC+TGCTGGGGTGG Identical overlapping sequence, SEQ ID NO: 375 SEQ ID NO: 376 ends differ miR-375 miR-375GSP CATGATCAGCTGGGCCAAGATCACGCGAGC miR-375RP TT+TG+TTCGTTCGGC Identical SEQ ID NO: 387 SEQ ID NO: 388 miR-376a miR-376aGSP3 CATGATCAGCTGGGCCAAGAACGTGGAT miR-376aRP2 A+TCGTAGA+GGAAAATCCAC one or more base pairs differ SEQ ID NO: 467 SEQ ID NO: 468 miR-378 miR-378GSP CATGATCAGCTGGGCCAAGAACACAGGACC miR-378RP C+TC+CTGACTCCAGG Identical SEQ ID NO: 391 SEQ ID NO: 392 miR-379 miR-379_GSP7 CATGATCAGCTGGGCCAAGATACGTTC miR-379RP2 T+GGT+AGACTATGGAACG Identical overlapping sequence, SEQ ID NO: 393 SEQ ID NO: 394 ends differ miR-380-5p miR-380-5pGSP CATGATCAGCTGGGCCAAGAGCGCATGTTC miR-380-5pRP T+GGT+TGACCATAGA Identical SEQ ID NO: 395 SEQ ID NO: 396 miR-380-3p miR-380-3pGSP CATGATCAGCTGGGCCAAGAAAGATGTGGA miR-380-3pRP TA+TG+TAGTATGGTCCACA one or more base pairs differ SEQ ID NO: 395 SEQ ID NO: 483 miR-381 miR-381GSP2 CATGATCAGCTGGGCCAAGAACAGAGAGC miR-381RP2 TATA+CAA+GGGCAAGCT Identical SEQ ID NO: 399 SEQ ID NO: 400 miR-382 miR-382GSP CATGATCAGCTGGGCCAAGACGAATCCACC miR-382RP G+AA+GTTGTTCGTGGT Identical SEQ ID NO: 401 SEQ ID NO: 402 miR-383 miR-383GSP CATGATCAGCTGGGCCAAGAAGCCACAGTC miR-383RP2 A+GATC+AGAAGGTGACTGT one or more base pairs differ SEQ ID NO: 465 SEQ ID NO: 466 miR-384 miR-384_GSP9 CATGATCAGCTGGGCCAAGATGTGAACAA miR-384_RP5 ATT+CCT+AG+AAATTGTTC one or more base pairs differ SEQ ID NO: 470 SEQ ID NO: 471 miR-410 miR-410 GSP9 CATGATCAGCTGGGCCAAGAACAGGCCAT miR-410RP AA+TA+TAA+CA+CAGATGGC Identical SEQ ID NO: 405 SEQ ID NO: 406 miR-412 miR-412 GSP10 CATGATCAGCTGGGCCAAGAACGGCTAGTG miR-412RP A+CTT+CACCTGGTCCACTA Identical SEQ ID NO: 407 SEQ ID NO: 408 miR-424 miR-424GSP CATGATCAGCTGGGCCAAGATCCAAAACAT miR-424RP2 C+AG+CAGCAATTCATGTTTT one or more base pairs differ SEQ ID NO: 474 SEQ ID NO: 414 miR-425 miR-425GSP CATGATCAGCTGGGCCAAGAGGCGGACACG miR-425RP A+TC+GGGAATGTCGT Identical SEQ ID NO: 417 SEQ ID NO: 418 miR-429 miR-429_GSP11 CATGATCAGCTGGGCCAAGAACGGCATTACC miR-429RP5 T+AATAC+TG+TCTGGTAATG one or more base pairs differ SEQ ID NO: 479 SEQ ID NO: 480 miR-431 miR-431 GSP10 CATGATCAGCTGGGCCAAGATGCATGACGG miR-431RP T+GT+CTTGCAGGCCG Identical overlapping sequence, SEQ ID NO: 421 SEQ ID NO: 422 ends differ miR-448 miR-448GSP CATGATCAGCTGGGCCAAGAATGGGACATC miR-448RP TTG+CATA+TGTAGGATG Identical SEQ ID NO: 423 SEQ ID NO: 424 miR-449 miR-449GSP10 CATGATCAGCTGGGCCAAGAACCAGCTAAC miR-449RP2 T+GG+CAGTGTATTGTTAGC Identical SEQ ID NO: 425 SEQ ID NO: 426 miR-450 miR-450GSP CATGATCAGCTGGGCCAAGATATTAGGAAC miR-450RP TTTT+TG+CGATGTGTT Identical SEQ ID NO: 427 SEQ ID NO: 428 miR-451 miR-451 GSP10 CATGATCAGCTGGGCCAAGAAAACTCAGTA miR-451RP AAA+CCG+TTA+CCATTACTGA Identical overlapping sequence, SEQ ID NO: 429 SEQ ID NO: 430 ends differ let7a let7a-GSP2 CATGATCAGCTGGGCCAAGAAACTATAC let7a-RP T+GA+GGTAGTAGGTTG Identical overlapping sequence, SEQ ID NO: 431 SEQ ID NO: 432 ends differ let7b let7b-GSP2 CATGATCAGCTGGGCCAAGAAACCACAC let7b-RP T+GA+GGTAGTAGGTTG Identical SEQ ID NO: 433 SEQ ID NO: 432 let7c let7c-GSP2 CATGATCAGCTGGGCCAAGAAACCATAC let7c-RP T+GA+GGTAGTAGGTTG Identical SEQ ID NO: 434 SEQ ID NO: 432 let7d let7d-GSP2 CATGATCAGCTGGGCCAAGAACTATGCA let7d-RP A+GA+GGTAGTAGGTTG Identical SEQ ID NO: 435 SEQ ID NO: 436 let7e let7e-GSP2 CATGATCAGCTGGGCCAAGAACTATACA let7e-RP T+GA+GGTAGGAGGTTG Identical SEQ ID NO: 437 SEQ ID NO: 438 let7f let7f-GSP2 CATGATCAGCTGGGCCAAGAAACTATAC let7f-RP T+GA+GGTAGTAGATTG Identical overlapping sequence, SEQ ID NO: 439 SEQ ID NO: 440 ends differ let7g let7g-GSP2 CATGATCAGCTGGGCCAAGAACTGTACA let7g-RP T+GA+GGTAGTAGTTTG Identical SEQ ID NO: 441 SEQ ID NO: 442 let7i let7i-GSP2 CATGATCAGCTGGGCCAAGAACAGCACA let7i-RP T+GA+GGTAGTAGTTTG Identical SEQ ID NO: 443 SEQ ID NO: 444
Example 5
[0156] This Example describes the detection and analysis of expression profiles for three microRNAs in total RNA isolated from twelve different tissues using methods in accordance with an embodiment of the present invention.
[0157] Methods: Quantitative analysis of miR-1, miR-124 and miR-150 microRNA templates was determined using 0.5 μg of First Choice total RNA (Ambion, Inc.) per 10 μl primer extension reaction isolated from the following tissues: brain, heart, intestine, kidney, liver, lung, lymph, ovary, skeletal muscle, spleen, thymus and uterus. The primer extension enzyme and quantitative PCR reactions were carried out as described above in EXAMPLE 3, using the following PCR primers:
miR-1 Template:
TABLE-US-00010 extension primer: (SEQ ID NO: 47) CATGATCAGCTGGGCCAAGATACATACTTC reverse primer: (SEQ ID NO: 48) T+G+GAA+TG+TAAAGAAGT forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA
miR-124 Template:
TABLE-US-00011 extension primer: (SEQ ID NO: 149) CATGATCAGCTGGGCCAAGATGGCATTCAC reverse primer: (SEQ ID NO: 150) T+TA+AGGCACGCGGT forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA
miR-150 template:
TABLE-US-00012 extension primer: (SEQ ID NO: 213) CATGATCAGCTGGGCCAAGACACTGGTA reverse primer: (SEQ ID NO: 214) T+CT+CCCAACCCTTG forward primer: (SEQ ID NO: 13) CATGATCAGCTGGGCCAAGA
[0158] Results. The expression profiles for miR-1, miR-124 and miR-150 are shown in FIGS. 3A, 3B, and 3C, respectively. The data in FIGS. 3A-3C are presented in units of microRNA copies per 10 pg of total RNA (y-axis). These units were chosen since human cell lines typically yield ≦10 pg of total RNA per cell. Hence the data shown are estimates of microRNA copies per cell. The numbers on the x-axis correspond to the following tissues: (1) brain, (2) heart, (3) intestine, (4) kidney, (5) liver, (6) lung, (7) lymph, (8) ovary, (9) skeletal muscle, (10) spleen, (11) thymus and (12) uterus.
[0159] Consistent with previous reports, very high levels of striated muscle-specific expression were found for miR-1 (as shown in FIG. 3A), and high levels of brain expression were found for miR-124 (as shown in FIG. 3B) (see Lagos-Quintana et al., RNA 9:175-179, 2003). Quantitative analysis reveals that these microRNAs are present at tens to hundreds of thousands of copies per cell. These data are in agreement with quantitative Northern blot estimates of miR-1 and miR-124 levels (see Lim et al., Nature 433:769-773, 2005). As shown in FIG. 3C, miR-150 was found to be highly expressed in the immune-related lymph node, thymus and spleen samples which is also consistent with previous findings (see Baskerville et al., RNA 11:241-247, 2005).
Example 6
[0160] This Example describes the selection and validation of primers for detecting mammalian microRNAs of interest.
[0161] Rationale: In order to perform multiple assays to detect a plurality of microRNA targets in a single sample (i.e., multiplex PCR), it is important that the assays work under uniform reverse transcriptase and PCR cycling conditions in a common buffer system with a single universal primer. The following primer design principles and high throughput assays were utilized to identify useful primer sets for desired microRNA targets that work well under the designated reaction conditions.
[0162] Primer Design:
[0163] As described in Example 2, the sensitivity of an assay to detect mammalian microRNA targets using the methods of the invention may be measured by the cycle threshold (Ct) value. The lower the Ct value (e.g., the fewer number of cycles), the more sensitive is the assay. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) (preferably in the range of from about 5 to about 25, such as from about 10 to about 20), and broad dynamic range (preferably in the range of from about 10 and 35, such as ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.
[0164] microRNA Target Templates: Representative mammalian microRNA target templates (h=human, r=rat, m=mouse) are provided in Table 9 (SEQ ID NO:966 to SEQ ID NO:1043) which are publicly available and accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the "miRBase sequence database" as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.
[0165] Extension Primers:
[0166] Empirical data generated as described in Examples 1-5 suggests that gene specific (GS) extension primers are primarily responsible for the dynamic range of the assays for detecting mammalian microRNA targets using the methods described herein. As described in Example 2, it was determined that the dynamic range (ΔCt) and specificity of the assays tested decreased for extension primers having gene specific regions below 6 to 7 nucleotides. Therefore, in order to optimize microRNA detection assays, extension primers were designed that have 7 to 10 nucleotide overlap with the microRNA target of interest. Exemplary extension primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8 (SEQ ID NO:500 to SEQ ID NO:965). These exemplary extension primers have a gene specific (GS) region from 7 to 10 nucleotide overlap with the microRNA target of interest.
[0167] Reverse Primers:
[0168] Unmodified and locked nucleic acid (LNA)-containing reverse primers were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). Based on the data generated as described in Examples 1-5, it was determined that the design of the reverse primers contributes to the efficiency of the PCR reactions, with the observation that the longer the reverse primer, the better the PCR performance. However, it was also observed that the longer the overlap with the extension primer, the higher the background. Therefore, the reverse primers were designed to be as long as possible while minimizing the overlap with the gene specific portion of the extension primer, in order to reduce the non-specific background signal.
[0169] In addition, as described in Example 3, LNA base substitutions may be selected to raise the predicted Tm of the primer, with two or three LNA base substitutions typically substituted within the first 8 nucleotides from the 5' end of the reverse primer oligonucleotide. Exemplary reverse primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8. While these exemplary reverse primers contain LNA base substitutions (the "+" symbol preceding a nucleotide designates an LNA substitution), this feature is optional and not required.
[0170] Selection and validation of primers for a desired target:
[0171] Assay oligonucleotide selection is made in two steps as follows:
[0172] 1) Primer designs were determined using the principles described above. Typically, 4 extension primer candidates and 2 reverse primer candidates were designed for each microRNA target of interest. The extension primers in each set overlap the gene specific region by 7, 8, 9 and 10 nucleotides, respectively, at the 3' end. Exemplary primers designed according to these design principles are provided in TABLE 8 for the microRNA targets listed in TABLE 9.
[0173] Assay design to validate the candidate primer sets (Assay #1)
[0174] microRNA Target:
[0175] Exemplary target microRNA miR-495 has an RNA target sequence (SEQ ID NO:966) that is conserved across human (h), mouse (m) and rat (r), as indicated by the designation "hmr"-miR-495 in TABLE 9. Therefore, the primer designed for this target sequence would be expected to be useful to detect miR-495 in samples obtained from human, mouse, and rat.
[0176] microRNA miR-495 target RNA sequence: 5' AAACAAACAUGGUGCACUUCUU 3' (SEQ ID NO:966)
TABLE-US-00013 Extension Primers (4 candidates) (SEQ ID NO: 500) hmr-miR-495GS10: 5' CATGATCAGCTGGGCCAAGAAAGAAGTGCA 3' (SEQ ID NO: 501) hmr-miR-495GS9: 5' CATGATCAGCTGGGCCAAGAAAGAAGTGC 3' (SEQ ID NO: 502) hmr-miR-495GS8: 5' CATGATCAGCTGGGCCAAGAAAGAAGTG 3' (SEQ ID NO: 503) hmr-miR-495GS7: 5' CATGATCAGCTGGGCCAAGAAAGAAGT Reverse Primers (2 candidates) (SEQ ID NO: 504) hmr-miR-495RP1: 5' AAA+CAAA+CA+TGGTGCAC 3' (SEQ ID NO: 505) hmr-miR-495RP2: 5' AAA+C+AAA+CATGGTGC 3'
[0177] 2) The primers designed as described above were tested to find pairs that showed both high sensitivity and high dynamic range in quantitative PCR assays, using the assay methods described in Example 2. The optimal combination of extension primer and reverse primer was determined for the target microRNA by testing all combinations of primers in the presence or absence of DNA template. It is preferable to use DNA rather than RNA template to test the oligo pairs because it is less likely to degrade than RNA. Degraded templates result in misleading assay data. Therefore, HPLC purified DNA template molecules are preferred.
[0178] TABLE 8 shows exemplary primer sets for use in detection assays for 78 microRNA targets (shown in TABLE 9). The candidate primers for use in these assays were designed to specifically detect human (h), mouse (m) and rat (r) microRNAs, or microRNAs from one or more species. For example, assays with the "hmr" prefix are designed to detect a perfectly conserved microRNA in all three species, whereas a "mr" prefix means the assay is designed to detect a microRNA conserved between mouse and rat, but not human. Nucleotides preceded by a plus (+) sign may be optionally locked (LNA). TABLE 9 shows the microRNA target sequence for each assay.
TABLE-US-00014 TABLE 8 EXEMPLARY PRIMER SETS FOR DETECTING MAMMALIAN MICRORNA TARGETS Extension Reverse Assay Number Target microRNA Primer Name Extension Primer Sequence Primer Name Reverse Primer Sequence Comments 1 hmr-miR-495 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTGCA Hmr-miR- AAA+CAAA+CA+TGGTGCAC Conserved across all 495GS10 SEQ ID NO: 500 495RP1 SEQ ID NO: 504 three species Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTGC Hmr-miR- AAA+C+AAA+CATGGTGC 495GS9 SEQ ID NO: 501 495RP2 SEQ ID NO: 505 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGTG 495GS8 SEQ ID NO: 502 Hmr-miR- CATGATCAGCTGGGCCAAGAAAGAAGT 495GS7 SEQ ID NO: 503 2 mr-miR-291a- mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACAAA mr-mIR-291a- AA+AG+TGCTTCCACTTTGT Mouse/rat specific; seed 3p 291a- SEQ ID NO: 506 3pRP1 SEQ ID NO: 510 region ortholog to human 3pGS10 miR-371/2 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACAA mr-mIR-291a- AA+AG+TG+CTTCCACTTT 291a-3pGS9 SEQ ID NO: 507 3pRP2 SEQ ID NO: 511 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACACA 291a-3pGS8 SEQ ID NO: 508 mr-mIR- CATGATCAGCTGGGCCAAGAGGCACAC 291a-3pGS7 SEQ ID NO: 509 3 m-miR-291b- m-mIR- CATGATCAGCTGGGCCAAGAGACAAACAAA m-mIR-291b- AA+AG+TG+CAT+CCATTTTGT Mouse specific; seed 3p 291b- SEQ ID NO: 512 3pRP1 SEQ ID NO: 516 region ortholog to human 3pGS10 miR-371/2 m-mIR- CATGATCAGCTGGGCCAAGAGACAAACAA m-mIR-291b- AA+AG+TG+CATCCATTTT 291b-3pGS9 SEQ ID NO: 513 3pRP2 SEQ ID NO: 517 m-mIR- CATGATCAGCTGGGCCAAGAGACAAACA 291b-3pGS8 SEQ ID NO: 514 m-mIR- CATGATCAGCTGGGCCAAGAGACAAAC 291b-3pGS7 SEQ ID NO: 515 4 h-miR-519a h-miR- CATGATCAGCTGGGCCAAGAGTAACACTCT h-miR- AA+AG+TG+CATCCTTTTAGAGT Human specific; 519aGS10 SEQ ID NO: 518 519aRP1 SEQ ID NO: 522 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAGTAACACTC h-miR- AA+AG+TG+CATCCTTTTAGA 519aGS9 SEQ ID NO: 519 519aRP2 SEQ ID NO: 523 h-miR- CATGATCAGCTGGGCCAAGAGTAACACT 519aGS8 SEQ ID NO: 520 h-miR- CATGATCAGCTGGGCCAAGAGTAACAC 519aGS7 SEQ ID NO: 521 5 h-miR-519b h-miR- CATGATCAGCTGGGCCAAGAAAACCTCTAA h-miR- AA+AG+TG+CATCCTTTTAG Human specific; 519bGS10 SEQ ID NO: 524 519bRP1 SEQ ID NO: 528 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAAACCTCTA h-miR- AA+AG+TG+CATCCTTTT 519bGS9 SEQ ID NO: 525 519bRP2 SEQ ID NO: 529 h-miR- CATGATCAGCTGGGCCAAGAAAACCTCT 519bGS8 SEQ ID NO: 526 h-miR- CATGATCAGCTGGGCCAAGAAAACCTC 519bGS7 SEQ ID NO: 527 6 h-miR-519c h-miR- CATGATCAGCTGGGCCAAGAATCCTCTAAA h-miR- AA+AG+TG+CATCTTTTTAGA Human specific; 519cGS10 SEQ ID NO: 530 519cRP1 SEQ ID NO: 534 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAATCCTCTAA h-miR- AA+AG+TG+CATCTTTTTA 519cGS9 SEQ ID NO: 531 519cRP2 SEQ ID NO: 535 h-miR- CATGATCAGCTGGGCCAAGAATCCTCTA 519cGS8 SEQ ID NO: 532 h-miR- CATGATCAGCTGGGCCAAGAATCCTCT 519cGS7 SEQ ID NO: 533 7 h-miR-519d h-miR- CATGATCAGCTGGGCCAAGAACACTCTAAA h-miR- C+AAAG+TGCCTCCCTTTAG Human specific; 519dGS10 SEQ ID NO: 536 519dRP1 SEQ ID NO: 540 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAACACTCTAA h-miR- C+AA+AG+TGCCTCCCTTT 519dGS9 SEQ ID NO: 537 519dRP2 SEQ ID NO: 541 h-miR- CATGATCAGCTGGGCCAAGAACACTCTA 519dGS8 SEQ ID NO: 538 h-miR- CATGATCAGCTGGGCCAAGAACACTCT 519dGS7 SEQ ID NO: 539 8 h-miR-520a h-miR- CATGATCAGCTGGGCCAAGAACAGTCCAAA h-miR- AA+AG+TGCTTCCCTTTGG Human specific; 520aGS10 SEQ ID NO: 542 520aRP1 SEQ ID NO: 546 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAACAGTCCAA h-miR- AA+AG+T+GCTTCCCTTT 520aGS9 SEQ ID NO: 543 520aRP2 SEQ ID NO: 547 h-miR- CATGATCAGCTGGGCCAAGAACAGTCCA 520aGS8 SEQ ID NO: 544 h-miR- CATGATCAGCTGGGCCAAGAACAGTCC 520aGS7 SEQ ID NO: 545 9 h-miR-520b h-miR- CATGATCAGCTGGGCCAAGACCCTCTAAAA h-miR- AA+AG+T+GCTTCCTTTTAG Human specific; 520bGS10 SEQ ID NO: 548 520bRP1 SEQ ID NO: 552 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGACCCTCTAAA h-miR- AA+AG+TG+CTTCCTTTTA 520bGS9 SEQ ID NO: 549 520bRP2 SEQ ID NO: 553 h-miR- CATGATCAGCTGGGCCAAGACCCTCTAA 520bGS8 SEQ ID NO: 550 h-miR- CATGATCAGCTGGGCCAAGACCCTCTA 520bGS7 SEQ ID NO: 551 10 h-miR-520d h-miR- CATGATCAGCTGGGCCAAGAAACCCACCAA h-miR- AA+AG+TGCTTCTCTTTGGT Human specific; 520dGS10 SEQ ID NO: 554 520dRP1 SEQ ID NO: 558 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAACCCACCA h-miR- AA+AG+TG+CTTCTCTTTG 520dGS9 SEQ ID NO: 555 520dRP2 SEQ ID NO: 559 h-miR- CATGATCAGCTGGGCCAAGAAACCCACC 520dGS8 SEQ ID NO: 556 h-miR- CATGATCAGCTGGGCCAAGAAACCCAC 520dGS7 SEQ ID NO: 557 11 h-miR-520e h-miR- CATGATCAGCTGGGCCAAGACCCTCAAAAA h-miR- AA+AG+TGCTTCCTTTTTG Human specific; 520eGS10 SEQ ID NO: 560 520eRP1 SEQ ID NO: 564 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGACCCTCAAAA h-miR- AA+AG+T+GCTTCCTTTTT 520eGS9 SEQ ID NO: 561 520eRP2 SEQ ID NO: 565 h-miR- CATGATCAGCTGGGCCAAGACCCTCAAA 520eGS8 SEQ ID NO: 562 h-miR- CATGATCAGCTGGGCCAAGACCCTCAA 520eGS7 SEQ ID NO: 563 12 h-miR-520f h-miR- CATGATCAGCTGGGCCAAGAAACCCTCTAA h-miR- A+AG+TGCTTCCTTTTAGA Human specific; 520fGS10 SEQ ID NO: 566 520fRP1 SEQ ID NO: 570 implicated in oncogenesis h-miR- CATGATCAGCTGGGCCAAGAAACCCTCTA h-miR- A+AG+T+GCTTCCTTTTA 520fGS9 SEQ ID NO: 567 520fRP2 SEQ ID NO: 571 h-miR- CATGATCAGCTGGGCCAAGAAACCCTCT 520fGS8 SEQ ID NO: 568 h-miR- CATGATCAGCTGGGCCAAGAAACCCTC 520fGS7 SEQ ID NO: 569 13 mr-miR-329 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGTTA mr-miR- AA+CA+CACCCAGCTAACC Specific for mouse/rat 329G510 SEQ ID NO: 572 329RP1 SEQ ID NO: 576 ortholog mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGTT mr-miR- AA+CA+CACCCAGCTAA 329GS9 SEQ ID NO: 573 329RP2 SEQ ID NO: 577 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGGT 329GS8 SEQ ID NO: 574 mr-miR- CATGATCAGCTGGGCCAAGAAAAAAGG 329GS7 SEQ ID NO: 575 14 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACCGA hmr-miR- AA+CATT+CATTGTTGTCGGT Conserved across all 181d 181dGS10 SEQ ID NO: 578 181dRP1 SEQ ID NO: 582 three species hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACCG hmr-miR- AA+CA+TT+CATTGTTGTCG 181dGS9 SEQ ID NO: 579 181dRP2 SEQ ID NO: 583 hmr-miR- CATGATCAGCTGGGCCAAGAAACCCACC 181dGS8 SEQ ID NO: 580 hmr-miR- CATGATCAGCTGGGCCAAGAAACCCAC 181dGS7 SEQ ID NO: 581 15 has-miR-193b hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGGAC hmr-miR- AA+CT+GGCCCTCAAAGTCCC Conserved across all 193bGS10 SEQ ID NO: 584 193bRP1 SEQ ID NO: 588 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGGA hmr-miR- AA+CT+GGCCCTCAAAGTC 193bGS9 SEQ ID NO: 585 193bRP2 SEQ ID NO: 589 hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGGG 193bGS8 SEQ ID NO: 586 hmr-miR- CATGATCAGCTGGGCCAAGAAAAGCGG 193bGS7 SEQ ID NO: 587 16 h-miR-362 h-miR- CATGATCAGCTGGGCCAAGAACTCACACCT h-miR-362RP1 AAT+CCTT+GGAACCTAGGTG Assay specific for human 362GS10 SEQ ID NO: 590 SEQ ID NO: 594 ortholog h-miR- CATGATCAGCTGGGCCAAGAACTCACACC h-miR-362RP2 AA+TC+CTT+GGAACCTAGG 362GS9 SEQ ID NO: 591 SEQ ID NO: 595 h-miR- CATGATCAGCTGGGCCAAGAACTCACAC 362GS8 SEQ ID NO: 592 h-miR- CATGATCAGCTGGGCCAAGAACTCACA 362GS7 SEQ ID NO: 593 17 mr-miR-362 mr-mIR- CATGATCAGCTGGGCCAAGATTCACACCTA mr-mIR-362- AA+TCCTT+GGAACCTAGGT Assay specific for rodent 362-3pGS10 SEQ ID NO: 596 3pRP1 SEQ ID NO: 600 ortholog mr-mIR- CATGATCAGCTGGGCCAAGATTCACACCT mr-mIR-362- AA+TC+CTT+GGAACCTAG 362-3pGS9 SEQ ID NO: 597 3pRP2 SEQ ID NO: 601 mr-mIR- CATGATCAGCTGGGCCAAGATTCACACC 362-3pGS8 SEQ ID NO: 598 mr-mIR- CATGATCAGCTGGGCCAAGATTCACAC 362-3pGS7 SEQ ID NO: 599 18 h-miR-500 h-miR- CATGATCAGCTGGGCCAAGACAGAATCCTT h-miR-500RP1 A+TG+CACCTGGGCAAGGA Assay specific for human 500GS10 SEQ ID NO: 602 SEQ ID NO: 606 ortholog h-miR- CATGATCAGCTGGGCCAAGACAGAATCCT h-miR-500RP2 A+TG+CACCTGGGCAAG 500GS9 SEQ ID NO: 603 SEQ ID NO: 607 h-miR- CATGATCAGCTGGGCCAAGACAGAATCC 500GS8 SEQ ID NO: 604 h-miR- CATGATCAGCTGGGCCAAGACAGAATC 500GS7 SEQ ID NO: 605 19 mmu-miR- mr-miR- CATGATCAGCTGGGCCAAGACTGAACCCTT mr-miR- A+TGCA+CCTGGGCAAGGG Assay specific for rodent 500 500GS10 SEQ ID NO: 608 500RP1 SEQ ID NO: 612 ortholog mr-miR- CATGATCAGCTGGGCCAAGACTGAACCCT mr-miR- A+TGCA+CCTGGGCAAG 500GS9 SEQ ID NO: 609 500RP2 SEQ ID NO: 613 mr-miR- CATGATCAGCTGGGCCAAGACTGAACCC 500GS8 SEQ ID NO: 610 mr-miR- CATGATCAGCTGGGCCAAGACTGAACC 500GS7 SEQ ID NO: 611 20 h-miR-501 h-miR- CATGATCAGCTGGGCCAAGATCTCACCCAG h-miR-501RP1 AA+T+CCTT+TGTCCCTGGG Assay specific for human 501GS10 SEQ ID NO: 614 SEQ ID NO: 618 ortholog h-miR- CATGATCAGCTGGGCCAAGATCTCACCCA h-miR-501RP2 AAT+CCTT+TGTCCCTGG 501GS9 SEQ ID NO: 615 SEQ ID NO: 619 h-miR- CATGATCAGCTGGGCCAAGATCTCACCC 501GS8 SEQ ID NO: 616 h-miR- CATGATCAGCTGGGCCAAGATCTCACC 501GS7 SEQ ID NO: 617 21 mr-miR-501 mr-miR- CATGATCAGCTGGGCCAAGATTTCACCCAG mr-miR- AA+T+CC+TTTGTCCCTGGG Assay specific for rodent 501GS10 SEQ ID NO: 620 501RP1 SEQ ID NO: 624 ortholog mr-miR- CATGATCAGCTGGGCCAAGATTTCACCCA mr-miR- AA+T+CC+TTTGTCCCTG 501GS9 SEQ ID NO: 621 501RP2 SEQ ID NO: 625 mr-miR- CATGATCAGCTGGGCCAAGATTTCACCC 501GS8 SEQ ID NO: 622 mr-miR- CATGATCAGCTGGGCCAAGATTTCACC 501GS7 SEQ ID NO: 623 22 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATGAC hmr-miR- AAT+CG+TACAGGGTCAT Conserved across all 487b 487bGS10 SEQ ID NO: 626 487bRP1 SEQ ID NO: 630 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATGA hmr-miR- A+AT+CG+TACAGGGTC 487bGS9 SEQ ID NO: 627 487bRP2 SEQ ID NO: 631 hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGATG 487bGS8 SEQ ID NO: 628 hmr-miR- CATGATCAGCTGGGCCAAGAAGTGGAT 487bGS7 SEQ ID NO: 629 23 h-miR-489 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGTAT h-miR-489RP1 AG+TGA+CATCACATATACG Assay specific for human 489GS10 SEQ ID NO: 632 SEQ ID NO: 636 ortholog h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGTA h-miR-489RP2 A+G+TGA+CATCACATATAC 489GS9 SEQ ID NO: 633 SEQ ID NO: 637 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCGT 489GS8 SEQ ID NO: 634 h-miR- CATGATCAGCTGGGCCAAGAGCTGCCG 489GS7 SEQ ID NO: 635 24 m-miR-489 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCATAT m-miR-489RP1 AATGA+CA+CCACATATATG Assay specific for mouse
489GS10 SEQ ID NO: 638 SEQ ID NO: 642 ortholog m-miR- CATGATCAGCTGGGCCAAGAGCTGCCATA m-miR-489RP2 AA+TGA+CA+CCACATAT 489GS9 SEQ ID NO: 639 SEQ ID NO: 643 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCAT 489GS8 SEQ ID NO: 640 m-miR- CATGATCAGCTGGGCCAAGAGCTGCCA 489GS7 SEQ ID NO: 641 25 r-miR-489 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCATAT r-miR-489RP1 AA+TGA+CA+TCACATATATG Assay specific for rat 489GS10 SEQ ID NO: 644 SEQ ID NO: 648 ortholog r-miR- CATGATCAGCTGGGCCAAGAGCTGCCATA r-miR-489RP2 AAT+GA+CA+TCACATATAT 489GS9 SEQ ID NO: 645 SEQ ID NO: 649 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCAT 489GS8 SEQ ID NO: 646 r-miR- CATGATCAGCTGGGCCAAGAGCTGCCA 489GS7 SEQ ID NO: 647 26 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGGAG hmr-miR-425- AA+TGA+CACGATCACTCCC Conserved across all 425-5p 425-5pGS10 SEQ ID NO: 650 5pRP1 SEQ ID NO: 654 three species hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGGA hmr-miR-425- AA+T+GA+CACGATCACTC 425-5pGS9 SEQ ID NO: 651 5pRP2 SEQ ID NO: 655 hmr-miR- CATGATCAGCTGGGCCAAGATCAACGGG 425-5pGS8 SEQ ID NO: 652 hmr-miR- CATGATCAGCTGGGCCAAGATCAACGG 425-5pGS7 SEQ ID NO: 653 27 hmr-miR-652 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAACCC hmr-miR- AAT+GGCGCCACTAGGGTT Conserved across all 652GS10 SEQ ID NO: 656 652RP1 SEQ ID NO: 660 three species hmr-miR- CATGATCAGCTGGGCCAAGATGCACAACC hmr-miR- AAT+GG+CGCCACTAGGG 652GS9 SEQ ID NO: 657 652RP2 SEQ ID NO: 661 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAAC 652GS8 SEQ ID NO: 658 hmr-miR- CATGATCAGCTGGGCCAAGATGCACAA 652GS7 SEQ ID NO: 659 28 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCATCA hmr-miR-485- AGA+GGCTGGCCGTGATG Conserved across all 485-5p 485-5pGS10 SEQ ID NO: 662 5pRP1 SEQ ID NO: 666 three species hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCATC hmr-miR-485- AGA+GGCTGGCCGTGA 485-5pGS9 SEQ ID NO: 663 5pRP2 SEQ ID NO: 667 hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCAT 485-5pGS8 SEQ ID NO: 664 hmr-miR- CATGATCAGCTGGGCCAAGAGAATTCA 485-5pGS7 SEQ ID NO: 665 29 has-miR-485- hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGAGA hmr-miR-485- AG+TCATA+CACGGCTCTCC Conserved across all 3p 485-3pGS10 SEQ ID NO: 668 3pRP1 SEQ ID NO: 672 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGAG hmr-miR-485- AG+TC+ATACACGGCTCT 485-3pGS9 SEQ ID NO: 669 3pRP2 SEQ ID NO: 673 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGGA 485-3pGS8 SEQ ID NO: 670 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGAGG 485-3pGS7 SEQ ID NO: 671 30 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACGAATATAAC hmr-miR-369- A+GA+TC+GACCGTGTTAT Conserved across all 369-5p 369-5pGS10 SEQ ID NO: 674 5pRP1 SEQ ID NO: 678 three species hmr-miR- CATGATCAGCTGGGCCAAGACGAATATAA hmr-miR-369- A+GA+TCGACCGTGTT 369-5pGS9 SEQ ID NO: 675 5pRP2 SEQ ID NO: 679 hmr-miR- CATGATCAGCTGGGCCAAGACGAATATA 369-5pGS8 SEQ ID NO: 676 hmr-miR- CATGATCAGCTGGGCCAAGACGAATAT 369-5pGS7 SEQ ID NO: 677 31 hmr-miR-671 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGCCC hmr-miR- A+GGAAGCCCTGGAGGGGCT Conserved across all 671GS10 SEQ ID NO: 680 671RP1 SEQ ID NO: 684 three species hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGCC hmr-miR- A+GGAAGCCCTGGAGGGG 671GS9 SEQ ID NO: 681 671RP2 SEQ ID NO: 685 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAGC 671GS8 SEQ ID NO: 682 hmr-miR- CATGATCAGCTGGGCCAAGACCTCCAG 671GS7 SEQ ID NO: 683 32 h-miR-449b h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTAAC h-miR- A+GGC+AGTGTATTGTTAG Assay specific for human 449bGS10 SEQ ID NO: 686 449bRP1 SEQ ID NO: 690 ortholog h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTAA h-miR- AG+GC+AG+TGTATTGTT 449bGS9 SEQ ID NO: 687 449bRP2 SEQ ID NO: 691 h-miR- CATGATCAGCTGGGCCAAGAGCCAGCTA 449bGS8 SEQ ID NO: 688 h-miR- CATGATCAGCTGGGCCAAGAGCCAGCT 449bGS7 SEQ ID NO: 689 33 mr-miR-449b mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAGCA mr-miR- A+GGC+AGTGCATTGCTA Assay specific for rodent 449bGS10 SEQ ID NO: 692 449bRP1 SEQ ID NO: 696 ortholog mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAGC mr-miR- A+GG+CAGTGCATTGC 449bGS9 SEQ ID NO: 693 449bRP2 SEQ ID NO: 697 mr-miR- CATGATCAGCTGGGCCAAGACCAGCTAG 449bGS8 SEQ ID NO: 694 mr-miR- CATGATCAGCTGGGCCAAGACCAGCTA 449bGS7 SEQ ID NO: 695 34 m-miR-699 m-miR- CATGATCAGCTGGGCCAAGACGAGCCAGGT m-miR-699RP1 A+GGCAGTGCGACCTG Mouse specific; ortholog 699GS10 SEQ ID NO: 698 SEQ ID NO: 702 to miR-34c m-miR- CATGATCAGCTGGGCCAAGACGAGCCAGG m-miR-699RP2 A+GG+CAGTGCGACC 699GS9 SEQ ID NO: 699 SEQ ID NO: 703 m-miR- CATGATCAGCTGGGCCAAGACGAGCCAG 699GS8 SEQ ID NO: 700 m-miR- CATGATCAGCTGGGCCAAGACGAGCCA 699GS7 SEQ ID NO: 701 35 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTGCT hmr-miR-409- A+GGT+TACCCGAGCAACT Conserved across all 409-5p 409-5pGS10 SEQ ID NO: 704 5pRP1 SEQ ID NO: 708 three species hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTGC hmr-miR-409- A+GG+TTACCCGAGCAA 409-5pGS9 SEQ ID NO: 705 5pRP2 SEQ ID NO: 709 hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTTG 409-5pGS8 SEQ ID NO: 706 hmr-miR- CATGATCAGCTGGGCCAAGACAAAGTT 409-5pGS7 SEQ ID NO: 707 36 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTTCA hmr-miR-409- G+AA+TGTTGCTCGGTGAAC Conserved across all 409-3p 409-3pGS10 SEQ ID NO: 710 3pRP1 SEQ ID NO: 714 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTTC hmr-miR-409- G+AA+TGTTGCTCGGTGA 409-3pGS9 SEQ ID NO: 711 3pRP2 SEQ ID NO: 715 hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGTT 409-3pGS8 SEQ ID NO: 712 hmr-miR- CATGATCAGCTGGGCCAAGAAAGGGGT 409-3pGS7 SEQ ID NO: 713 37 hmr-miR-491 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGGAA hmr-miR- AG+TGG+GGAACCCTTCCA Conserved across all 491GS10 SEQ ID NO: 716 491RP1 SEQ ID NO: 720 three species hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGGA hmr-miR- AG+TG+GGGAACCCTTC 491GS9 SEQ ID NO: 717 491RP2 SEQ ID NO: 721 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATGG 491GS8 SEQ ID NO: 718 hmr-miR- CATGATCAGCTGGGCCAAGACCTCATG 491GS7 SEQ ID NO: 719 38 h-miR-384 h-miR- CATGATCAGCTGGGCCAAGATATGAACAAT h-miR-384RP1 A+TT+CCT+AGAAATTGTTC Assay specific for human 384GS10 SEQ ID NO: 722 SEQ ID NO: 726 ortholog h-miR- CATGATCAGCTGGGCCAAGATATGAACAA h-miR-384RP2 A+TT+CCT+AG+AAATTGT 384GS9 SEQ ID NO: 723 SEQ ID NO: 727 h-miR- CATGATCAGCTGGGCCAAGATATGAACA 384GS8 SEQ ID NO: 724 h-miR- CATGATCAGCTGGGCCAAGATATGAAC 384GS7 SEQ ID NO: 725 39 mr-miR-384 mr-miR- CATGATCAGCTGGGCCAAGATGTGAACAAT mr-miR- A+TT+CCT+AGAAATTGTT Assay specific for rodent 384GS10 SEQ ID NO: 728 384RP1 SEQ ID NO: 732 ortholog mr-miR- CATGATCAGCTGGGCCAAGATGTGAACAA mr-miR- A+TT+CCT+AG+AAATTGTT 384GS9 SEQ ID NO: 729 384RP2 SEQ ID NO: 733 mr-miR- CATGATCAGCTGGGCCAAGATGTGAACA 384GS8 SEQ ID NO: 730 mr-miR- CATGATCAGCTGGGCCAAGATGTGAAC 384GS7 SEQ ID NO: 731 40 hmr-miR-20b hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCACTA hmr-miR- C+AA+AG+TGCTCATAGTGCA Conserved across all 20bGS10 SEQ ID NO: 734 20bRP1 SEQ ID NO: 738 three species hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCACT hmr-miR- CAA+AG+TG+CTCATAGTG 20bGS9 SEQ ID NO: 735 20bRP2 SEQ ID NO: 739 hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCAC 20bGS8 SEQ ID NO: 736 hmr-miR- CATGATCAGCTGGGCCAAGAACCTGCA 20bGS7 SEQ ID NO: 737 41 hmr-miR-490 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGGAG hmr-miR- C+AA+CCTGGAGGACTCCA Conserved across all 490GS10 SEQ ID NO: 740 490RP1 SEQ ID NO: 744 three species hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGGA hmr-miR- CAA+CCT+GGAGGACTC 490GS9 SEQ ID NO: 741 490RP2 SEQ ID NO: 745 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATGG 490GS8 SEQ ID NO: 742 hmr-miR- CATGATCAGCTGGGCCAAGACAGCATG 490GS7 SEQ ID NO: 743 42 hmr-miR-497 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCACA hmr-miR- C+AG+CAGCACACTGTGG Conserved across all 497GS10 SEQ ID NO: 746 497RP1 SEQ ID NO: 750 three species hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCAC hmr-miR- C+AG+CAGCACACTGTG 497GS9 SEQ ID NO: 747 497RP2 SEQ ID NO: 751 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACCA 497GS8 SEQ ID NO: 748 hmr-miR- CATGATCAGCTGGGCCAAGAACAAACC 497GS7 SEQ ID NO: 749 43 h-miR-301b h-miR- CATGATCAGCTGGGCCAAGATGCTTTGACA h-miR- C+AG+TG+CAATGATATTGTCA Assay specific for human 301bGS10 SEQ ID NO: 752 301bRP1 SEQ ID NO: 756 ortholog h-miR- CATGATCAGCTGGGCCAAGATGCTTTGAC h-miR- C+AG+TG+CAATGATATTGT 301bGS9 SEQ ID NO: 753 301bRP2 SEQ ID NO: 757 h-miR- CATGATCAGCTGGGCCAAGATGCTTTGA 301bGS8 SEQ ID NO: 754 h-miR- CATGATCAGCTGGGCCAAGATGCTTTG 301bGS7 SEQ ID NO: 755 44 mr-miR-301b mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGACA mr-miR- C+AG+TG+CAATGGTATTGTCA Assay specific for rodent 301bGS10 SEQ ID NO: 758 301bRP1 SEQ ID NO: 762 ortholog mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGAC mr-miR- C+AG+TG+CAATGGTATTGT 301bGS9 SEQ ID NO: 759 301bRP2 SEQ ID NO: 763 mr-miR- CATGATCAGCTGGGCCAAGATGCTTTGA 301bGS8 SEQ ID NO: 760 mr-miR- CATGATCAGCTGGGCCAAGATGCTTTG 301bGS7 SEQ ID NO: 761 45 hmr-miR-721 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCTTT hmr-miR- C+AG+TG+CAATTAAAAGGG Conserved across all 721GS10 SEQ ID NO: 764 721RP1 SEQ ID NO: 768 three species hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCTT hmr-miR- C+AG+TG+CAATTAAAAG 721GS9 SEQ ID NO: 765 721RP2 SEQ ID NO: 769 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCCT 721GS8 SEQ ID NO: 766 hmr-miR- CATGATCAGCTGGGCCAAGATTCCCCC 721GS7 SEQ ID NO: 767 46 hmr-miR-532 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCTAC hmr-miR- CA+TG+CCTTGAGTGTAGG Conserved across all 532GS10 SEQ ID NO: 770 532RP1 SEQ ID NO: 774 three species hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCTA hmr-miR- CA+TG+CCTTGAGTGTA 532GS9 SEQ ID NO: 771 532RP2 SEQ ID NO: 775 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCCT 532GS8 SEQ ID NO: 772 hmr-miR- CATGATCAGCTGGGCCAAGAACGGTCC 532GS7 SEQ ID NO: 773 47 h-miR-488 h-miR- CATGATCAGCTGGGCCAAGATTGAGAGTGC h-miR-488RP1 C+CCA+GATAATGGCACT Assay specific for human 488GS10 SEQ ID NO: 776 SEQ ID NO: 780 ortholog h-miR- CATGATCAGCTGGGCCAAGATTGAGAGTG h-miR-488RP2 C+CC+A+GATAATGGCA 488GS9 SEQ ID NO: 777 SEQ ID NO: 781 h-miR- CATGATCAGCTGGGCCAAGATTGAGAGT 488GS8 SEQ ID NO: 778 h-miR- CATGATCAGCTGGGCCAAGATTGAGAG 488GS7 SEQ ID NO: 779 48 mr-miR-488 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGTGC mr-miR- C+CCA+GATAATAGCACT Assay specific for rodent 488GS10 SEQ ID NO: 782 488RP1 SEQ ID NO: 786 ortholog mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGTG mr-miR- C+CC+A+GATAATAGCA 488GS9 SEQ ID NO: 783 488RP2 SEQ ID NO: 787 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAGT 488GS8 SEQ ID NO: 784 mr-miR- CATGATCAGCTGGGCCAAGATTGAGAG 488GS7 SEQ ID NO: 785 49 hmr-miR-539 hmr-miR- CATGATCAGCTGGGCCAAGAACACACCAAG hmr-miR- GG+AG+AAATTATCCTTGGT Conserved across all 539GS10 SEQ ID NO: 788 539RP1 SEQ ID NO: 792 three species
hmr-miR- CATGATCAGCTGGGCCAAGAACACACCAA hmr-miR- G+GA+G+AAATTATCCTTGG 539GS9 SEQ ID NO: 789 539RP2 SEQ ID NO: 793 hmr-miR- CATGATCAGCTGGGCCAAGAACACACCA 539GS8 SEQ ID NO: 790 hmr-miR- CATGATCAGCTGGGCCAAGAACACACC 539GS7 SEQ ID NO: 791 50 h-miR-505 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAACCA h-miR-505RP1 GT+CAA+CACTTGCTGGTT Assay specific for human 505GS10 SEQ ID NO: 794 SEQ ID NO: 798 ortholog h-miR- CATGATCAGCTGGGCCAAGAGAGGAAACC h-miR-505RP2 G+T+CAA+CACTTGCTGG 505GS9 SEQ ID NO: 795 SEQ ID NO: 799 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAAC 505GS8 SEQ ID NO: 796 h-miR- CATGATCAGCTGGGCCAAGAGAGGAAA 505GS7 SEQ ID NO: 797 51 mr-miR-505 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCAGC mr-miR- CG+T+CAA+CA+CTTGCTGGT Assay specific for rodent 505GS10 SEQ ID NO: 800 505RP1 SEQ ID NO: 804 ortholog mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCAG mr-miR- CG+T+CAA+CA+CTTGCTG 505GS9 SEQ ID NO: 801 505RP2 SEQ ID NO: 805 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACCA 505GS8 SEQ ID NO: 802 mr-miR- CATGATCAGCTGGGCCAAGAGGAAACC 505GS7 SEQ ID NO: 803 52 h-miR-18b h-miR- CATGATCAGCTGGGCCAAGATAACTGCACT h-miR-18bRP1 TAA+GG+TGCATCTAGTGC Assay specific for human 18bGS10 SEQ ID NO: 806 SEQ ID NO: 810 ortholog h-miR- CATGATCAGCTGGGCCAAGATAACTGCAC h-miR-18bRP2 T+AA+GG+TGCATCTAGT 18bGS9 SEQ ID NO: 807 SEQ ID NO: 811 h-miR- CATGATCAGCTGGGCCAAGATAACTGCA 18bGS8 SEQ ID NO: 808 h-miR- CATGATCAGCTGGGCCAAGATAACTGC 18bGS7 SEQ ID NO: 809 53 mr-miR-18b mr-miR- CATGATCAGCTGGGCCAAGATAACAGCACT mr-miR- T+AA+GG+TGCATCTAGTGC Assay specific for rodent 18bGS10 SEQ ID NO: 812 18bRP1 SEQ ID NO: 816 ortholog mr-miR- CATGATCAGCTGGGCCAAGATAACAGCAC mr-miR- TAA+GG+TG+CATCTAGT 18bGS9 SEQ ID NO: 813 18bRP2 SEQ ID NO: 817 mr-miR- CATGATCAGCTGGGCCAAGATAACAGCA 18bGS8 SEQ ID NO: 814 mr-miR- CATGATCAGCTGGGCCAAGATAACAGC 18bGS7 SEQ ID NO: 815 54 hmr-miR-503 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTGTT hmr-miR- T+AGC+AGCGGGAACAGT Conserved across all 503GS10 SEQ ID NO: 818 503RP1 SEQ ID NO: 822 three species hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTGT hmr-miR- T+AGC+AGCGGGAACA 503GS9 SEQ ID NO: 819 503RP2 SEQ ID NO: 823 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACTG 503GS8 SEQ ID NO: 820 hmr-miR- CATGATCAGCTGGGCCAAGACAGTACT 503GS7 SEQ ID NO: 821 55 hmr-miR-455 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAGTC hmr-miR- TA+TG+TGCCTTTGGACTA Conserved across all 455GS10 SEQ ID NO: 824 455RP1 SEQ ID NO: 828 three species hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAGT hmr-miR- TA+TG+TGCCTTTGGAC 455GS9 SEQ ID NO: 825 455RP2 SEQ ID NO: 829 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTAG 455GS8 SEQ ID NO: 826 hmr-miR- CATGATCAGCTGGGCCAAGACGATGTA 455GS7 SEQ ID NO: 827 56 hmr-miR-92b hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGGGA hmr-miR- TAT+TG+CACTCGTCCCG Conserved across all 92bGS10 SEQ ID NO: 830 92bRP1 SEQ ID NO: 834 three species hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGGG hmr-miR- TAT+TG+CACTCGTCCC 92bGS9 SEQ ID NO: 831 92bRP2 SEQ ID NO: 835 hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCGG 92bGS8 SEQ ID NO: 832 hmr-miR- CATGATCAGCTGGGCCAAGAGAGGCCG 92bGS7 SEQ ID NO: 833 57 h-miR-483 h-miR- CATGATCAGCTGGGCCAAGAAGAAGACGGG h-miR-483RP1 T+CAC+TCCTCTCCTCCCGT Assay specific for human 483GS10 SEQ ID NO: 836 SEQ ID NO: 840 ortholog h-miR- CATGATCAGCTGGGCCAAGAAGAAGACGG h-miR-483RP2 T+CAC+TCCTCTCCTCCC 483GS9 SEQ ID NO: 837 SEQ ID NO: 841 h-miR- CATGATCAGCTGGGCCAAGAAGAAGACG 483GS8 SEQ ID NO: 838 h-miR- CATGATCAGCTGGGCCAAGAAGAAGAC 483GS7 SEQ ID NO: 839 58 mr-miR-483 mr-miR- CATGATCAGCTGGGCCAAGAACAAGACGGG mr-miR- TC+ACTCCTCCCCTCCCGT Assay specific for rodent 483GS10 SEQ ID NO: 842 483RP1 SEQ ID NO: 846 ortholog mr-miR- CATGATCAGCTGGGCCAAGAACAAGACGG mr-miR- TC+ACTCCTCCCCTCCC 483GS9 SEQ ID NO: 843 483RP2 SEQ ID NO: 847 mr-miR- CATGATCAGCTGGGCCAAGAACAAGACG 483GS8 SEQ ID NO: 844 mr-miR- CATGATCAGCTGGGCCAAGAACAAGAC 483GS7 SEQ ID NO: 845 59 hmr-miR-484 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAGGG hmr-miR- TCA+GGCTCAGTCCCCTC Conserved across all 484GS10 SEQ ID NO: 848 484RP1 SEQ ID NO: 852 three species hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAGG hmr-miR- TC+AGGCTCAGTCCCC 484GS9 SEQ ID NO: 849 484RP2 SEQ ID NO: 853 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGAG 484GS8 SEQ ID NO: 850 hmr-miR- CATGATCAGCTGGGCCAAGAATCGGGA 484GS7 SEQ ID NO: 851 60 mmu-miR- hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCAAA hmr-miR- TC+CCTGAGGAGCCCTTTGA Rodent specific; ortholog 351 351GS10 SEQ ID NO: 854 351RP1 SEQ ID NO: 858 to human miR-125 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCAA hmr-miR- TC+CCTGAGGAGCCCTTT 351GS9 SEQ ID NO: 855 351RP2 SEQ ID NO: 859 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTCA 351GS8 SEQ ID NO: 856 hmr-miR- CATGATCAGCTGGGCCAAGACAGGCTC 351GS7 SEQ ID NO: 857 61 hmr-miR-615 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAGAC hmr-miR- TC+CGAGCCTGGGTCTC Conserved across all 615GS10 SEQ ID NO: 860 615RP1 SEQ ID NO: 864 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAGA hmr-miR- TC+CGAGCCTGGGTC 615GS9 SEQ ID NO: 861 615RP2 SEQ ID NO: 865 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGAG 615GS8 SEQ ID NO: 862 hmr-miR- CATGATCAGCTGGGCCAAGAAGAGGGA 615GS7 SEQ ID NO: 863 62 hmr-miR-486 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGCAG hmr-miR- T+CC+TGTACTGAGCTGCC Conserved across all 486GS10 SEQ ID NO: 866 486RP1 SEQ ID NO: 870 three species hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGCA hmr-miR- T+CC+TGTACTGAGCTG 486GS9 SEQ ID NO: 867 486RP2 SEQ ID NO: 871 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGGC 486GS8 SEQ ID NO: 868 hmr-miR- CATGATCAGCTGGGCCAAGACTCGGGG 486GS7 SEQ ID NO: 869 63 hmr-miR-494 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCCCG hmr-miR- T+GA+AA+CATACACGGGA Conserved across all 494GS10 SEQ ID NO: 872 494RP1 SEQ ID NO: 876 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCCC hmr-miR- T+GA+AA+CATACACGG 494GS9 SEQ ID NO: 873 494RP2 SEQ ID NO: 877 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTCC 494GS8 SEQ ID NO: 874 hmr-miR- CATGATCAGCTGGGCCAAGAAGGTTTC 494GS7 SEQ ID NO: 875 64 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACACA hmr-miR-493- T+GAA+GGTCTACTGTG Conserved across all 493-3p 493-3pGS10 SEQ ID NO: 878 3pRP1 SEQ ID NO: 882 three species hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACAC hmr-miR-493- T+GAA+GGTCTACTGT 493-3pGS9 SEQ ID NO: 879 3pRP2 SEQ ID NO: 883 hmr-miR- CATGATCAGCTGGGCCAAGACTGGCACA 493-3pGS8 SEQ ID NO: 880 hmr-miR- CATGATCAGCTGGGCCAAGACTGGCAC 493-3pGS7 SEQ ID NO: 881 65 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATGGA hmr-miR- T+GA+GAAC+TGAATTCCATA Conserved across all 146b 146bGS10 SEQ ID NO: 884 146bRP1 SEQ ID NO: 888 three species hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATGG hmr-miR- T+GA+GAAC+TGAATTCCA 146bGS9 SEQ ID NO: 885 146bRP2 SEQ ID NO: 889 hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTATG 146bGS8 SEQ ID NO: 886 hmr-miR- CATGATCAGCTGGGCCAAGAAGCCTAT 146bGS7 SEQ ID NO: 887 66 r-miR-1 r-miR- CATGATCAGCTGGGCCAAGATACACACTTC r-miR-1RP1 T+G+GAA+TGTAAAGAAGTG Assay specific for rat 1GS10 SEQ ID NO: 890 SEQ ID NO: 894 ortholog r-miR-1GS9 CATGATCAGCTGGGCCAAGATACACACTT r-miR-1RP2 T+G+GAA+TGTAAAGAAG SEQ ID NO: 891 SEQ ID NO: 895 r-miR-1GS8 CATGATCAGCTGGGCCAAGATACACACT SEQ ID NO: 892 r-miR-1GS7 CATGATCAGCTGGGCCAAGATACACAC SEQ ID NO: 893 67 h-miR-675-5p h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGGGC h-miR-675- T+GGTGCGGAGAGGGCCCA Assay specific for human 5pGS10 SEQ ID NO: 896 5pRP1 SEQ ID NO: 900 ortholog h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGGG h-miR-675- T+GGTGCGGAGAGGGC 5pGS9 SEQ ID NO: 897 5pRP2 SEQ ID NO: 901 h-miR-675- CATGATCAGCTGGGCCAAGACACTGTGG 5pGS8 SEQ ID NO: 898 h-miR-675- CATGATCAGCTGGGCCAAGACACTGTG 5pGS7 SEQ ID NO: 899 68 mr-miR-675- mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGGCC mr-miR-675- T+GGTGCGGAAAGGGCC Assay specific for rodent 5p 675-5pGS10 SEQ ID NO: 902 5pRP1 SEQ ID NO: 906 ortholog mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGGC mr-miR-675- T+GGTGCGGAAAGGG 675-5pGS9 SEQ ID NO: 903 5pRP2 SEQ ID NO: 907 mr-miR- CATGATCAGCTGGGCCAAGAACTGTGGG 675-5pGS8 SEQ ID NO: 904 mr-miR- CATGATCAGCTGGGCCAAGAACTGTGG 675-5pGS7 SEQ ID NO: 905 69 hmr-miR-668 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGGCC hmr-miR- TG+TCACTCGGCTCGGCC Conserved across all 668GS10 SEQ ID NO: 908 668RP1 SEQ ID NO: 912 three species hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGGC hmr-miR- TG+TCACTCGGCTCGG 668GS9 SEQ ID NO: 909 668RP2 SEQ ID NO: 913 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGGG 668GS8 SEQ ID NO: 910 hmr-miR- CATGATCAGCTGGGCCAAGAGTAGTGG 668GS7 SEQ ID NO: 911 70 r-miR-346 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAGGC r-miR-346RP1 TGTC+TGCCTGAGTGCCTG Assay specific for rat 346GS10 SEQ ID NO: 914 SEQ ID NO: 918 ortholog r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAGG r-miR-346RP2 TGTC+TGCCTGAGTGCC 346GS9 SEQ ID NO: 915 SEQ ID NO: 919 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCAG 346GS8 SEQ ID NO: 916 r-miR- CATGATCAGCTGGGCCAAGAAGAGGCA 346GS7 SEQ ID NO: 917 71 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTATC hmr-miR-542- TG+TGA+CAGATTGATAACT Conserved across all 542-3p 542-3pGS10 SEQ ID NO: 920 3pRP1 SEQ ID NO: 924 three species hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTAT hmr-miR-542- TG+T+GA+CAGATTGATAA 542-3pGS9 SEQ ID NO: 921 3pRP2 SEQ ID NO: 925 hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTTA 542-3pGS8 SEQ ID NO: 922 hmr-miR- CATGATCAGCTGGGCCAAGATTCAGTT 542-3pGS7 SEQ ID NO: 923 72 hmr-miR- hmr-miR- CATGATCAGCTGGGCCAAGACGTGACATGATG hmr-miR-542- CTC+GG+GGATCATCATG Conserved across all 542-5p 542-5pGS10 SEQ ID NO: 926 5pRP1 SEQ ID NO: 930 three species hmr-miR- CATGATCAGCTGGGCCAAGACGTGACATG hmr-miR-542- C+TC+GGGGATCATCAT 542-5pGS9 SEQ ID NO: 927 5pRP2 SEQ ID NO: 931 hmr-miR- CATGATCAGCTGGGCCAAGACGTGACAT 542-5pGS8 SEQ ID NO: 928 hmr-miR- CATGATCAGCTGGGCCAAGACGTGACA 542-5pGS7 SEQ ID NO: 929 73 hmr-miR-499 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCACT hmr-miR- T+TAA+GA+CTTGCAGTGAT Conserved across all 499G510 SEQ ID NO: 932 499RP1 SEQ ID NO: 936 three species hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCAC hmr-miR- T+TAA+GA+CTTGCAGTG 499GS9 SEQ ID NO: 933 499RP2 SEQ ID NO: 937 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATCA 499GS8 SEQ ID NO: 934 hmr-miR- CATGATCAGCTGGGCCAAGAAAACATC 499GS7 SEQ ID NO: 935 74 hmr-miR-758 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGGAC hmr-miR- TT+TG+TGACCTGGTCCAC Conserved across all 758GS10 SEQ ID NO: 938 758RP1 SEQ ID NO: 942 three species hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGGA hmr-miR- TT+TG+T+GACCTGGTCC
758GS9 SEQ ID NO: 939 758RP2 SEQ ID NO: 943 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTGG 758GS8 SEQ ID NO: 940 hmr-miR- CATGATCAGCTGGGCCAAGAGTTAGTG 758GS7 SEQ ID NO: 941 75 hmr-miR-194 miR- CATGATCAGCTGGGCCAAGATCCACATGGA miR-194RP1 TG+TAA+CAGCAACTCCA Conserved across all 194GSP10 SEQ ID NO: 944 SEQ ID NO: 948 three species miR- CATGATCAGCTGGGCCAAGATCCACATGG miR-RP2 TG+TAA+CA+GCAACTCCAT 194GSP9 SEQ ID NO: 945 SEQ ID NO: 949 miR- CATGATCAGCTGGGCCAAGATCCACATG 194GSP8 SEQ ID NO: 946 miR- CATGATCAGCTGGGCCAAGATCCACAT 194GSP7 SEQ ID NO: 947 76 hmr-miR-206 mir- CATGATCAGCTGGGCCAAGACCACACACTT mir-206RP1 T+GGAA+TGTAAGGAAGT Conserved across all 206GSP10 SEQ ID NO: 950 SEQ ID NO: 954 three species mir- CATGATCAGCTGGGCCAAGACCACACACT miR-206RP2 T+G+GAA+TGTAAGGAAGTGT 206GSP9 SEQ ID NO: 951 SEQ ID NO: 955 mir- CATGATCAGCTGGGCCAAGACCACACAC 206GSP8 SEQ ID NO: 952 mir- CATGATCAGCTGGGCCAAGACCACACA 206GSP7 SEQ ID NO: 953 77 hmr-miR-1 miR-1GS10 CATGATCAGCTGGGCCAAGATACATACTTC miR-1RP1 TG+GAA+TG+TAAAGAAGTA Conserved across all (SEQ ID NO: 47) (SEQ ID NO: 959) three species miR-1GS9 CATGATCAGCTGGGCCAAGATACATACTT (SEQ miR-1RP2 T+G+GAA+TG+TAAAGAAGT ID NO: 956) (SEQ ID NO: 48) miR-1GS8 CATGATCAGCTGGGCCAAGATACATACT (SEQ ID NO: 957) miR-1GS7 CATGATCAGCTGGGCCAAGATACATAC (SEQ ID NO: 958) 78 hmr-miR-9 miR-9GS10 CATGATCAGCTGGGCCAAGATCATACAGCT miR-9RP1 T+CTTT+GGTTATCTAGCT (SEQ Conserved across all (SEQ ID NO: 960) ID NO: 964) three species miR-9G59 CATGATCAGCTGGGCCAAGATCATACAGC (SEQ miR-9RP2 TC+TTT+GGTT+ATCTAGCTGTA ID NO: 961) (SEQ ID NO: 965) miR-9G58 CATGATCAGCTGGGCCAAGATCATACAG (SEQ ID NO: 962) miR-9G57 CATGATCAGCTGGGCCAAGATCATACA (SEQ ID NO: 963)
TABLE-US-00015 TABLE 9 SEQ Assay Target ID Number MicroRNA Name RNA target sequence NO: 1. hmr-miR-495 AAACAAACAUGGUGCACUUCUU 966 2. mr-miR-291a- AAAGUGCUUCCACUUUGUGUGCC 967 3p 3. m-mIR-291b-3p AAAGUGCAUCCAUUUUGUUUGUC 968 4. h-miR-519a AAAGUGCAUCCUUUUAGAGUGUUAC 969 5. h-miR-519b AAAGUGCAUCCUUUUAGAGGUUU 970 6. h-miR-519c AAAGUGCAUCUUUUUAGAGGAU 971 7. h-miR-519d CAAAGUGCCUCCCUUUAGAGUGU 972 8. h-miR-520a AAAGUGCUUCCCUUUGGACUGU 973 9. h-miR-520b AAAGUGCUUCCUUUUAGAGGG 974 10. h-miR-520d AAAGUGCUUCUCUUUGGUGGGUU 975 11. h-miR-520e AAAGUGCUUCCUUUUUGAGGG 976 12. h-miR-520f AAGUGCUUCCUUUUAGAGGGUU 977 13. mr-miR-329 AACACACCCAGCUAACCUUUUU 978 14. hmr-miR-181d AACAUUCAUUGUUGUCGGUGGGUU 979 15. hmr-miR-193b AACUGGCCCUCAAAGUCCCGCUUU 980 16. h-miR-362 AAUCCUUGGAACCUAGGUGUGAGU 981 17. mr-mIR-362-3p AAUCCUUGGAACCUAGGUGUGAA 982 18. h-miR-500 AUGCACCUGGGCAAGGAUUCUG 983 19. mr-miR-500 AUGCACCUGGGCAAGGGUUCAG 984 20. h-miR-501 AAUCCUUUGUCCCUGGGUGAGA 985 21. mr-miR-501 AAUCCUUUGUCCCUGGGUGAAA 986 22. hmr-miR-487b AAUCGUACAGGGUCAUCCACU 987 23. h-miR-489 AGUGACAUCACAUAUACGGCAGC 988 24. m-miR-489 AAUGACACCACAUAUAUGGCAGC 989 25. r-miR-489 AAUGACAUCACAUAUAUGGCAGC 990 26. hmr-miR-425- AAUGACACGAUCACUCCCGUUGA 991 5p 27. hmr-miR-652 AAUGGCGCCACUAGGGUUGUGCA 992 28. hmr-miR-485 AGAGGCUGGCCGUGAUGAAUUC 993 -5p 29. hmr-miR-485 AGUCAUACACGGCUCUCCUCUCU 994 -3p 30. hmr-miR-369 AGAUCGACCGUGUUAUAUUCG 995 -5p 31. hmr-miR-671 AGGAAGCCCUGGAGGGGCUGGAGG 996 32. h-miR-449b AGGCAGUGUAUUGUUAGCUGGC 997 33. mr-miR-449b AGGCAGUGCAUUGCUAGCUGG 998 34. m-miR-699 AGGCAGUGCGACCUGGCUCG 999 35. hmr-miR-409- AGGUUACCCGAGCAACUUUGCA 1000 5p 36. hmr-miR-409- GAAUGUUGCUCGGUGAACCCCUU 1001 3p 37. hmr-miR-491 AGUGGGGAACCCUUCCAUGAGG 1002 38. h-miR-384 AUUCCUAGAAAUUGUUCAUA 1003 39. mr-miR-384 AUUCCUAGAAAUUGUUCACA 1004 40. hmr-miR-20b CAAAGUGCUCAUAGUGCAGGUAG 1005 41. hmr-miR-490 CAACCUGGAGGACUCCAUGCUG 1006 42. hmr-miR-497 CAGCAGCACACUGUGGUUUGU 1007 43. h-miR-301b CAGUGCAAUGAUAUUGUCAAAGCA 1008 44. mr-miR-301b CAGUGCAAUGGUAUUGUCAAAGCA 1009 45. hmr-miR-721 CAGUGCAAUUAAAAGGGGGAA 1010 46. hmr-miR-532 CAUGCCUUGAGUGUAGGACCGU 1011 47. h-miR-488 CCCAGAUAAUGGCACUCUCAA 1012 48. mr-miR-488 CCCAGAUAAUAGCACUCUCAA 1013 49. hmr-miR-539 GGAGAAAUUAUCCUUGGUGUGU 1014 50. h-miR-505 GUCAACACUUGCUGGUUUCCUC 1015 51. mr-miR-505 CGUCAACACUUGCUGGUUUUCU 1016 52. h-miR-18b UAAGGUGCAUCUAGUGCAGUUA 1017 53. mr-miR-18b UAAGGUGCAUCUAGUGCUGUUA 1018 54. hmr-miR-503 UAGCAGCGGGAACAGUACUGC 1019 55. hmr-miR-455 UAUGUGCCUUUGGACUACAUCG 1020 56. hmr-miR-92b UAUUGCACUCGUCCCGGCCUC 1021 57. h-miR-483 UCACUCCUCUCCUCCCGUCUUCU 1022 58. mr-miR-483 UCACUCCUCCCCUCCCGUCUUGU 1023 59. hmr-miR-484 UCAGGCUCAGUCCCCUCCCGAU 1024 60. hmr-miR-351 UCCCUGAGGAGCCCUUUGAGCCUG 1025 61. hmr-miR-615 UCCGAGCCUGGGUCUCCCUCU 1026 62. hmr-miR-486 UCCUGUACUGAGCUGCCCCGAG 1027 63. hmr-miR-494 UGAAACAUACACGGGAAACCU 1028 64. hmr-miR-493- UGAAGGUCUACUGUGUGCCAG 1029 3p 65. hmr-miR-146b UGAGAACUGAAUUCCAUAGGCU 1030 66. r-miR-1 UGGAAUGUAAAGAAGUGUGUA 1031 67. h-miR-675-5p UGGUGCGGAGAGGGCCCACAGUG 1032 68. mr-miR-675-5p UGGUGCGGAAAGGGCCCACAGU 1033 69. hmr-miR-668 UGUCACUCGGCUCGGCCCACUAC 1034 70. r-miR-346 UGUCUGCCUGAGUGCCUGCCUCU 1035 71. hmr-miR-542- UGUGACAGAUUGAUAACUGAAA 1036 3p 72. hmr-miR-542- CUCGGGGAUCAUCAUGUCACG 1037 5p 73. hmr-miR-499 UUAAGACUUGCAGUGAUGUUU 1038 74. hmr-miR-758 UUUGUGACCUGGUCCACUAACC 1039 75. hmiR-194 UGUAACAGCAACUCCAUGUGGA 1040 76. hmiR-206 UGGAAUGUAAGGAAGUGUGUGG 1041 77. hmiR-1 UGGAAUGUAAAGAAGUAUGUA 1042 78. hmiR-9 UCUUUGGUUAUCUAGCUGUAUGA 1043
[0179] Assay Format:
[0180] Several candidate primer sets shown above in TABLE 8 were tested in a high-throughput assay testing format as follows:
[0181] Each test assay (e.g., assay #75, #76, #77 and #78 listed in TABLE 8) was run in 4×4 wells of a 96 well plate, with 6 assays per 96 well plate, thereby allowing for rapid determination of the optimal primer pair for each target.
[0182] For each assay, each of the 4 candidate extension (GS) primers were tested in a separate row of the 96 well plate. Each of the 2 reverse primers were tested plus (1 nM DNA) or minus template (10 mM Tris pH 7.6, 0.1 mM EDTA, 100 ng/ul yeast total RNA).
[0183] Following reverse transcription, one set of duplicate non-template control and template samples was tested against reverse primer 1 (RP1) and the other against reverse primer 2 (RP2).
[0184] Reverse Transcriptase Assay Conditions: [0185] 6 μl of RT master mix was added to all 96 wells [0186] 2 μl of 0.5 μM GS primers was added to four successive wells [0187] yeast RNA in TE (10 mM Tris pH 7.6, 0.1 mM EDTA) was added to all odd-numbered wells and pre-diluted DNA templates was added to even-numbered wells
[0188] Samples were mixed well and the reverse transcriptase step was carried out, followed by dilution with 80 μl TE (10 mM Tris pH 7.6, 0.1 mM EDTA).
[0189] 2 μl of the reverse transcription mixture was transferred into quadruplicate wells of a 384 well PCR plate preloaded with 80 PCR mix per well containing universal primer plus the appropriate reverse primers.
[0190] The quantitative PCR reaction results were evaluated on a real-time PCR instrument compatible with 384 well plates.
[0191] Ct values for the PCR reactions were determined based on a baseline threshold of 0.01. The sensitivity (Ct value of 1 nM template) and dynamic range (Ct of no-template control minus the Ct of the 1 nM template) were determined for each primer pair in each assay. The results of exemplary assays #75, #76, #77 and #78, listed in TABLE 8, are shown in TABLE 10 below.
TABLE-US-00016 TABLE 10 ASSAY RESULTS USING CANDIDATE PRIMER SETS FOR DETECTING MIR-1, MIR-9; MIR-194 AND MIR-206 Selected microRNA Dynamic for use in target Extension primer Reverse primer Sensitivity Range profiling miR-9 miR-9GS10 miR-9 RP1 13 9 - (SEQ ID NO: 1043) (SEQ ID NO: 960) (SEQ ID NO: 964) miR-9GS9 miR-9 RP1 13 4 - (SEQ ID NO: 961) (SEQ ID NO: 964) miR-9GS8 miR-9 RP1 10 0 - (SEQ ID NO:962) (SEQ ID NO: 964) miR-9GS7 miR-9 RP1 16 8 - (SEQ ID NO: 963) (SEQ ID NO: 964) miR-9GS10 miR-9 RP2 13 5 - (SEQ ID NO: 960) (SEQ ID NO: 965) miR-9GS9 miR-9 RP2 14 4 - (SEQ ID NO: 961) (SEQ ID NO: 965) miR-9GS8 miR-9 RP2 10 0 - (SEQ ID NO: 962) (SEQ ID NO: 965) miR-9GS7 miR-9 RP2 17 8 - (SEQ ID NO: 963) (SEQ ID NO: 965) miR-194 miR-194GS10 miR-194RP1 9 6 - (SEQ ID NO: 1040) (SEQ ID NO: 944) (SEQ ID NO: 948) miR-194GS9 miR-194RP1 11 5 - (SEQ ID NO: 945) (SEQ ID NO: 948) miR-194GS8 miR-194RP1 13 17 + (SEQ ID NO: 946) (SEQ ID NO: 948) miR-194GS7 miR-194RP1 15 17 - (SEQ ID NO: 947) (SEQ ID NO: 948) miR-194GS10 miR-194RP2 10 6 - (SEQ ID NO: 944) (SEQ ID NO: 949) miR-194GS9 miR-194RP2 11 6 - (SEQ ID NO: 945) (SEQ ID NO: 949) miR-194GS8 miR-194RP2 13 16 - (SEQ ID NO: 946) (SEQ ID NO: 949) miR-194GS7 miR-194RP2 17 16 - (SEQ ID NO: 947) (SEQ ID NO: 949) miR-1 miR-1 GS10 miR-1 RP1 15 15 - (SEQ ID NO: 1042) (SEQ ID NO: 47) (SEQ ID NO: 959) miR-1 GS9 miR-1 RP1 17 8 - (SEQ ID NO: 956) (SEQ ID NO: 959) miR-1 GS8 miR-1 RP1 19 11 - (SEQ ID NO: 957) (SEQ ID NO: 959) miR-1 GS7 miR-1 RP1 22 11 - (SEQ ID NO: 958) (SEQ ID NO: 959) miR-1 GS10 miR-1 RP2 13 15 + (SEQ ID NO: 47) (SEQ ID NO: 48) miR-1 GS9 miR-1 RP2 15 8 - (SEQ ID NO: 956) (SEQ ID NO: 48) miR-1 GS8 miR-1 RP2 17 11 - (SEQ ID NO: 957) (SEQ ID NO: 48) miR-1 GS7 miR-1 RP2 19 10 - (SEQ ID NO: 958) (SEQ ID NO: 48) miR-206 miR-206 GS10 miR-206RP1 15 10 - (SEQ ID NO: 1041) (SEQ ID NO: 950) (SEQ ID NO: 954) miR-206 GS9 miR-206RP1 16 10 - (SEQ ID NO: 951) (SEQ ID NO: 954) miR-206 GS8 miR-206RP1 17 14 - (SEQ ID NO: 952) (SEQ ID NO: 954) miR-206 GS7 miR-206RP1 20 20 - (SEQ ID NO: 953) (SEQ ID NO: 954) miR-206 GS10 miR-206RP2 10 8 - (SEQ ID NO: 950) (SEQ ID NO: 955) miR-206 GS9 miR-206RP2 11 9 - (SEQ ID NO: 951) (SEQ ID NO: 955) miR-206 GS8 miR-206RP2 11 11 - (SEQ ID NO: 952) (SEQ ID NO: 955) miR-206 GS7 miR-206RP2 13 20 + (SEQ ID NO: 953) (SEQ ID NO: 955)
[0192] Optimal primer pairs were identified based on superior sensitivity (e.g., a preferred range between 5 and 25) and dynamic range (e.g., a preferred range between 10 and 35) characteristics. As shown above in TABLE 10, an optimal primer pair was identified for miR-194: GS8 (SEQ ID NO:946) and RP1 (SEQ ID NO:948) with a sensitivity of 13 and a dynamic range of 17. An optimal primer pair was identified for miR-1: GS10 (SEQ ID NO:47) and RP2 (SEQ ID NO:48) with a sensitivity of 13 and a dynamic range of 15. An optimal primer pair was identified for miR-206: GS7 (SEQ ID NO:953) and RP2 (SEQ ID NO:955) with a sensitivity of 13 and a dynamic range of 20. As also shown in TABLE 10, the GS primers control specificity, as shown by the significant increase in dynamic range (driven by a decrease in background) in going from GS9 to GS8 (see, e.g., miR-194).
[0193] Candidate primers designed based on the principles described above, such as the additional exemplary primers listed in TABLE 8, or other candidate primers designed using the design principles described herein, may be tested using the screening methods described above. The assays may be further optimized by using HPLC purified templates to avoid problems associated with degraded templates.
[0194] It has also been determined that microRNAs that differ from each other in sequence by only 1, 2 or 3 nucleotide changes can be readily distinguished from one another through the use of the primers designed according to the design principles and methods described herein.
[0195] While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
Sequence CWU
1
1043120DNAArtificial SequencePrimer 1catgatcagc tgggccaaga
20233DNAArtificial SequencePrimer
2catgatcagc tgggccaaga aactatacaa cct
33331DNAArtificial SequencePrimer 3catgatcagc tgggccaaga tacatacttc t
31434DNAArtificial SequencePrimer
4catgatcagc tgggccaaga cacaaaccat tatg
34535DNAArtificial SequencePrimer 5catgatcagc tgggccaaga cgccaatatt tacgt
35631DNAArtificial SequencePrimer
6catgatcagc tgggccaaga tcaacatcag t
31732DNAArtificial SequencePrimer 7catgatcagc tgggccaaga ctgttcctgc tg
32836DNAArtificial SequencePrimer
8catgatcagc tgggccaaga acaaacacca ttgtca
36936DNAArtificial SequencePrimer 9catgatcagc tgggccaaga tggcattcac
cgcgtg 361032DNAArtificial SequencePrimer
10catgatcagc tgggccaaga tgagctacag tg
321136DNAArtificial SequencePrimer 11catgatcagc tgggccaaga aagggattcc
tgggaa 361233DNAArtificial SequencePrimer
12catgatcagc tgggccaaga cccctatcac gat
331320DNAArtificial SequencePrimer 13catgatcagc tgggccaaga
201415DNAArtificial SequencePrimer
14tgaggtagta ggttg
151517DNAArtificial SequencePrimer 15tggaatgtaa agaagta
171615DNAArtificial SequencePrimer
16tagcagcaca taatg
151714DNAArtificial SequencePrimer 17tagcagcacg taaa
141817DNAArtificial SequencePrimer
18tagcttatca gactgat
171914DNAArtificial SequencePrimer 19tggctcagtt cagc
142013DNAArtificial SequencePrimer
20tggagtgtga caa
132112DNAArtificial SequencePrimer 21ttaaggcacg cg
122215DNAArtificial SequencePrimer
22tgagatgaag cactg
152314DNAArtificial SequencePrimer 23gtccagtttt ccca
142416DNAArtificial SequencePrimer
24ttaatgctaa tcgtga
162532DNAArtificial SequencePrimer 25catgatcagc tgggccaaga gccaatattt ct
322631DNAArtificial SequencePrimer
26catgatcagc tgggccaaga gccaatattt c
312730DNAArtificial SequencePrimer 27catgatcagc tgggccaaga gccaatattt
302829DNAArtificial SequencePrimer
28catgatcagc tgggccaaga gccaatatt
292928DNAArtificial SequencePrimer 29catgatcagc tgggccaaga gccaatat
283027DNAArtificial SequencePrimer
30catgatcagc tgggccaaga gccaata
273126DNAArtificial SequencePrimer 31catgatcagc tgggccaaga gccaat
263225DNAArtificial SequencePrimer
32catgatcagc tgggccaaga gccaa
253324DNAArtificial SequencePrimer 33catgatcagc tgggccaaga gcca
243423DNAArtificial SequencePrimer
34catgatcagc tgggccaaga gcc
233532DNAArtificial SequencePrimer 35catgatcagc tgggccaaga gtctgtcaat tc
323631DNAArtificial SequencePrimer
36catgatcagc tgggccaaga gtctgtcaat t
313730DNAArtificial SequencePrimer 37catgatcagc tgggccaaga gtctgtcaat
303829DNAArtificial SequencePrimer
38catgatcagc tgggccaaga gtctgtcaa
293928DNAArtificial SequencePrimer 39catgatcagc tgggccaaga gtctgtca
284027DNAArtificial SequencePrimer
40catgatcagc tgggccaaga gtctgtc
274126DNAArtificial SequencePrimer 41catgatcagc tgggccaaga gtctgt
264225DNAArtificial SequencePrimer
42catgatcagc tgggccaaga gtctg
254324DNAArtificial SequencePrimer 43catgatcagc tgggccaaga gtct
244423DNAArtificial SequencePrimer
44catgatcagc tgggccaaga gtc
234515DNAArtificial SequencePrimer 45tagcagcaca gaaat
154615DNAArtificial SequencePrimer
46atgacctatg aattg
154730DNAArtificial SequencePrimer 47catgatcagc tgggccaaga tacatacttc
304816DNAArtificial SequencePrimer
48tggaatgtaa agaagt
164930DNAArtificial SequencePrimer 49catgatcagc tgggccaaga caacaaaatc
305018DNAArtificial SequencePrimer
50tggaagacta gtgatttt
185130DNAArtificial SequencePrimer 51catgatcagc tgggccaaga actttcggtt
305216DNAArtificial SequencePrimer
52taaagctaga taaccg
165330DNAArtificial SequencePrimer 53catgatcagc tgggccaaga cacaaattcg
305415DNAArtificial SequencePrimer
54taccctgtag atccg
155531DNAArtificial SequencePrimer 55catgatcagc tgggccaaga acaaattcgg t
315616DNAArtificial SequencePrimer
56taccctgtag aaccga
165730DNAArtificial SequencePrimer 57catgatcagc tgggccaaga cacaaaccat
305815DNAArtificial SequencePrimer
58tagcagcaca taatg
155929DNAArtificial SequencePrimer 59catgatcagc tgggccaaga tgtaaacca
296014DNAArtificial SequencePrimer
60tagcagcaca tcat
146129DNAArtificial SequencePrimer 61catgatcagc tgggccaaga cgccaatat
296214DNAArtificial SequencePrimer
62tagcagcacg taaa
146330DNAArtificial SequencePrimer 63catgatcagc tgggccaaga acaagtgcct
306414DNAArtificial SequencePrimer
64actgcagtga aggc
146529DNAArtificial SequencePrimer 65catgatcagc tgggccaaga actacctgc
296616DNAArtificial SequencePrimer
66caaagtgctt acagtg
166729DNAArtificial SequencePrimer 67catgatcagc tgggccaaga tcagttttg
296815DNAArtificial SequencePrimer
68tgtgcaaatc tatgc
156930DNAArtificial SequencePrimer 69catgatcagc tgggccaaga tcagttttgc
307014DNAArtificial SequencePrimer
70tgtgcaaatc catg
147128DNAArtificial SequencePrimer 71catgatcagc tgggccaaga ctacctgc
287218DNAArtificial SequencePrimer
72taaagtgctt atagtgca
187329DNAArtificial SequencePrimer 73catgatcagc tgggccaaga tcaacatca
297418DNAArtificial SequencePrimer
74tagcttatca gactgatg
187530DNAArtificial SequencePrimer 75catgatcagc tgggccaaga ggaaatccct
307614DNAArtificial SequencePrimer
76atcacattgc cagg
147730DNAArtificial SequencePrimer 77catgatcagc tgggccaaga ggtaatccct
307814DNAArtificial SequencePrimer
78atcacattgc cagg
147930DNAArtificial SequencePrimer 79catgatcagc tgggccaaga tcagaccgag
308015DNAArtificial SequencePrimer
80cattgcactt gtctc
158129DNAArtificial SequencePrimer 81catgatcagc tgggccaaga gcctatcct
298217DNAArtificial SequencePrimer
82ttcaagtaat ccaggat
178329DNAArtificial SequencePrimer 83catgatcagc tgggccaaga aacctatcc
298417DNAArtificial SequencePrimer
84ttcaagtaat tcaggat
178530DNAArtificial SequencePrimer 85catgatcagc tgggccaaga gcggaactta
308614DNAArtificial SequencePrimer
86ttcacagtgg ctaa
148730DNAArtificial SequencePrimer 87catgatcagc tgggccaaga gcagaactta
308814DNAArtificial SequencePrimer
88ttcacagtgg ctaa
148930DNAArtificial SequencePrimer 89catgatcagc tgggccaaga ctcaatagac
309014DNAArtificial SequencePrimer
90aaggagctca cagt
149128DNAArtificial SequencePrimer 91catgatcagc tgggccaaga aaccgatt
289216DNAArtificial SequencePrimer
92tagcaccatc tgaaat
169329DNAArtificial SequencePrimer 93catgatcagc tgggccaaga aacactgat
299419DNAArtificial SequencePrimer
94tagcaccatt tgaaatcag
199530DNAArtificial SequencePrimer 95catgatcagc tgggccaaga cttccagtcg
309616DNAArtificial SequencePrimer
96tgtaaacatc ctcgac
169730DNAArtificial SequencePrimer 97catgatcagc tgggccaaga agctgagtgt
309817DNAArtificial SequencePrimer
98tgtaaacatc ctacact
179930DNAArtificial SequencePrimer 99catgatcagc tgggccaaga gctgagagtg
3010017DNAArtificial SequencePrimer
100tgtaaacatc ctacact
1710130DNAArtificial SequencePrimer 101catgatcagc tgggccaaga cttccagtcg
3010214DNAArtificial SequencePrimer
102tgtaaacatc cccg
1410329DNAArtificial SequencePrimer 103catgatcagc tgggccaaga gctgtaaac
2910417DNAArtificial SequencePrimer
104ctttcagtcg gatgttt
1710530DNAArtificial SequencePrimer 105catgatcagc tgggccaaga tccagtcaag
3010616DNAArtificial SequencePrimer
106tgtaaacatc cttgac
1610730DNAArtificial SequencePrimer 107catgatcagc tgggccaaga cagctatgcc
3010814DNAArtificial SequencePrimer
108ggcaagatgc tggc
1410930DNAArtificial SequencePrimer 109catgatcagc tgggccaaga gcaacttagt
3011017DNAArtificial SequencePrimer
110tattgcacat tactaag
1711129DNAArtificial SequencePrimer 111catgatcagc tgggccaaga caatgcaac
2911215DNAArtificial SequencePrimer
112gtgcattgta gttgc
1511330DNAArtificial SequencePrimer 113catgatcagc tgggccaaga aacaaccagc
3011414DNAArtificial SequencePrimer
114tggcagtgtc ttag
1411530DNAArtificial SequencePrimer 115catgatcagc tgggccaaga caatcagcta
3011614DNAArtificial SequencePrimer
116taggcagtgt catt
1411730DNAArtificial SequencePrimer 117catgatcagc tgggccaaga gcaatcagct
3011814DNAArtificial SequencePrimer
118aggcagtgta gtta
1411930DNAArtificial SequencePrimer 119catgatcagc tgggccaaga caggccggga
3012015DNAArtificial SequencePrimer
120tattgcactt gtccc
1512130DNAArtificial SequencePrimer 121catgatcagc tgggccaaga ctacctgcac
3012214DNAArtificial SequencePrimer
122aaagtgctgt tcgt
1412330DNAArtificial SequencePrimer 123catgatcagc tgggccaaga tgctcaataa
3012419DNAArtificial SequencePrimer
124ttcaacgggt atttattga
1912530DNAArtificial SequencePrimer 125catgatcagc tgggccaaga gcaaaaatgt
3012614DNAArtificial SequencePrimer
126tttggcacta gcac
1412730DNAArtificial SequencePrimer 127catgatcagc tgggccaaga aacaatacaa
3012815DNAArtificial SequencePrimer
128tgaggtagta agttg
1512930DNAArtificial SequencePrimer 129catgatcagc tgggccaaga cacaagatcg
3013014DNAArtificial SequencePrimer
130aacccgtaga tccg
1413130DNAArtificial SequencePrimer 131catgatcagc tgggccaaga cgcaaggtcg
3013214DNAArtificial SequencePrimer
132cacccgtaga accg
1413330DNAArtificial SequencePrimer 133catgatcagc tgggccaaga cacaagttcg
3013414DNAArtificial SequencePrimer
134aacccgtaga tccg
1413530DNAArtificial SequencePrimer 135catgatcagc tgggccaaga cttcagttat
3013618DNAArtificial SequencePrimer
136tacagtactg tgataact
1813730DNAArtificial SequencePrimer 137catgatcagc tgggccaaga tcatagccct
3013814DNAArtificial SequencePrimer
138agcagcattg taca
1413930DNAArtificial SequencePrimer 139catgatcagc tgggccaaga acaggagtct
3014015DNAArtificial SequencePrimer
140tcaaatgctc agact
1514130DNAArtificial SequencePrimer 141catgatcagc tgggccaaga gctacctgca
3014216DNAArtificial SequencePrimer
142aaaagtgctt acagtg
1614330DNAArtificial SequencePrimer 143catgatcagc tgggccaaga atctgcactg
3014415DNAArtificial SequencePrimer
144taaagtgctg acagt
1514528DNAArtificial SequencePrimer 145catgatcagc tgggccaaga tgatagcc
2814615DNAArtificial SequencePrimer
146agcagcattg tacag
1514730DNAArtificial SequencePrimer 147catgatcagc tgggccaaga acaaacacca
3014814DNAArtificial SequencePrimer
148tggagtgtga caat
1414930DNAArtificial SequencePrimer 149catgatcagc tgggccaaga tggcattcac
3015014DNAArtificial SequencePrimer
150ttaaggcacg cggt
1415130DNAArtificial SequencePrimer 151catgatcagc tgggccaaga cacaggttaa
3015214DNAArtificial SequencePrimer
152tccctgagac cctt
1415330DNAArtificial SequencePrimer 153catgatcagc tgggccaaga tcacaagtta
3015414DNAArtificial SequencePrimer
154tccctgagac ccta
1415530DNAArtificial SequencePrimer 155catgatcagc tgggccaaga gcattattac
3015614DNAArtificial SequencePrimer
156tcgtaccgtg agta
1415728DNAArtificial SequencePrimer 157catgatcagc tgggccaaga cgcgtacc
2815819DNAArtificial SequencePrimer
158cattattact tttggtacg
1915930DNAArtificial SequencePrimer 159catgatcagc tgggccaaga agccaagctc
3016014DNAArtificial SequencePrimer
160tcggatccgt ctga
1416130DNAArtificial SequencePrimer 161catgatcagc tgggccaaga aaaagagacc
3016214DNAArtificial SequencePrimer
162tcacagtgaa ccgg
1416330DNAArtificial SequencePrimer 163catgatcagc tgggccaaga gaaagagacc
3016414DNAArtificial SequencePrimer
164tcacagtgaa ccgg
1416530DNAArtificial SequencePrimer 165catgatcagc tgggccaaga gcaagcccag
3016614DNAArtificial SequencePrimer
166ctttttgcgg tctg
1416730DNAArtificial SequencePrimer 167catgatcagc tgggccaaga atgccctttt
3016817DNAArtificial SequencePrimer
168cagtgcaatg ttaaaag
1716930DNAArtificial SequencePrimer 169catgatcagc tgggccaaga atgccctttc
3017014DNAArtificial SequencePrimer
170cagtgcaatg atga
1417130DNAArtificial SequencePrimer 171catgatcagc tgggccaaga cgaccatggc
3017215DNAArtificial SequencePrimer
172taacagtcta cagcc
1517330DNAArtificial SequencePrimer 173catgatcagc tgggccaaga acagctggtt
3017414DNAArtificial SequencePrimer
174ttggtcccct tcaa
1417530DNAArtificial SequencePrimer 175catgatcagc tgggccaaga tagctggttg
3017614DNAArtificial SequencePrimer
176ttggtcccct tcaa
1417730DNAArtificial SequencePrimer 177catgatcagc tgggccaaga ccctctggtc
3017814DNAArtificial SequencePrimer
178tgtgactggt tgac
1417930DNAArtificial SequencePrimer 179catgatcagc tgggccaaga tcacatagga
3018017DNAArtificial SequencePrimer
180tatggctttt tattcct
1718130DNAArtificial SequencePrimer 181catgatcagc tgggccaaga cacataggaa
3018216DNAArtificial SequencePrimer
182tatggctttt cattcc
1618330DNAArtificial SequencePrimer 183catgatcagc tgggccaaga tccatcatca
3018418DNAArtificial SequencePrimer
184actccatttg ttttgatg
1818530DNAArtificial SequencePrimer 185catgatcagc tgggccaaga ctacgcgtat
3018618DNAArtificial SequencePrimer
186tattgcttaa gaatacgc
1818729DNAArtificial SequencePrimer 187catgatcagc tgggccaaga cggcctgat
2918814DNAArtificial SequencePrimer
188agctggtgtt gtga
1418930DNAArtificial SequencePrimer 189catgatcagc tgggccaaga agacacgtgc
3019014DNAArtificial SequencePrimer
190tctacagtgc acgt
1419130DNAArtificial SequencePrimer 191catgatcagc tgggccaaga ctaccatagg
3019214DNAArtificial SequencePrimer
192agtggtttta ccct
1419329DNAArtificial SequencePrimer 193catgatcagc tgggccaaga ccatcttta
2919416DNAArtificial SequencePrimer
194taacactgtc tggtaa
1619528DNAArtificial SequencePrimer 195catgatcagc tgggccaaga tccataaa
2819616DNAArtificial SequencePrimer
196tgtagtgttt cctact
1619728DNAArtificial SequencePrimer 197catgatcagc tgggccaaga tgagctac
2819815DNAArtificial SequencePrimer
198tgagatgaag cactg
1519929DNAArtificial SequencePrimer 199catgatcagc tgggccaaga ctagtacat
2920016DNAArtificial SequencePrimer
200tacagtatag atgatg
1620129DNAArtificial SequencePrimer 201catgatcagc tgggccaaga aagggattc
2920214DNAArtificial SequencePrimer
202gtccagtttt ccca
1420328DNAArtificial SequencePrimer 203catgatcagc tgggccaaga aacccatg
2820416DNAArtificial SequencePrimer
204tgagaactga attcca
1620530DNAArtificial SequencePrimer 205catgatcagc tgggccaaga gcagaagcat
3020614DNAArtificial SequencePrimer
206gtgtgtggaa atgc
1420729DNAArtificial SequencePrimer 207catgatcagc tgggccaaga acaaagttc
2920818DNAArtificial SequencePrimer
208tcagtgcact acagaact
1820929DNAArtificial SequencePrimer 209catgatcagc tgggccaaga acaaagttc
2921014DNAArtificial SequencePrimer
210tcagtgcatc acag
1421129DNAArtificial SequencePrimer 211catgatcagc tgggccaaga ggagtgaag
2921214DNAArtificial SequencePrimer
212tctggctccg tgtc
1421328DNAArtificial SequencePrimer 213catgatcagc tgggccaaga cactggta
2821414DNAArtificial SequencePrimer
214tctcccaacc cttg
1421529DNAArtificial SequencePrimer 215catgatcagc tgggccaaga cctcaagga
2921615DNAArtificial SequencePrimer
216actagactga agctc
1521729DNAArtificial SequencePrimer 217catgatcagc tgggccaaga cccaagttc
2921814DNAArtificial SequencePrimer
218tcagtgcatg acag
1421929DNAArtificial SequencePrimer 219catgatcagc tgggccaaga tcacttttg
2922016DNAArtificial SequencePrimer
220ttgcatagtc acaaaa
1622129DNAArtificial SequencePrimer 221catgatcagc tgggccaaga aataggtca
2922217DNAArtificial SequencePrimer
222aatcatacac ggttgac
1722329DNAArtificial SequencePrimer 223catgatcagc tgggccaaga cgaaggcaa
2922415DNAArtificial SequencePrimer
224taggttatcc gtgtt
1522528DNAArtificial SequencePrimer 225catgatcagc tgggccaaga cccctatc
2822620DNAArtificial SequencePrimer
226ttaatgctaa tcgtgatagg
2022729DNAArtificial SequencePrimer 227catgatcagc tgggccaaga actcaccga
2922816DNAArtificial SequencePrimer
228aacattcaac gctgtc
1622929DNAArtificial SequencePrimer 229catgatcagc tgggccaaga actcaccga
2923016DNAArtificial SequencePrimer
230aacattcaac ctgtcg
1623130DNAArtificial SequencePrimer 231catgatcagc tgggccaaga tagttggcaa
3023215DNAArtificial SequencePrimer
232tggttctaga cttgc
1523329DNAArtificial SequencePrimer 233catgatcagc tgggccaaga tgtgagttc
2923414DNAArtificial SequencePrimer
234tttggcaatg gtag
1423529DNAArtificial SequencePrimer 235catgatcagc tgggccaaga cagtgaatt
2923614DNAArtificial SequencePrimer
236tatggcactg gtag
1423729DNAArtificial SequencePrimer 237catgatcagc tgggccaaga acccttatc
2923814DNAArtificial SequencePrimer
238tggacggaga actg
1423929DNAArtificial SequencePrimer 239catgatcagc tgggccaaga aagcccaaa
2924019DNAArtificial SequencePrimer
240caaagaattc tccttttgg
1924130DNAArtificial SequencePrimer 241catgatcagc tgggccaaga cggctgcaac
3024214DNAArtificial SequencePrimer
242tcgtgtcttg tgtt
1424330DNAArtificial SequencePrimer 243catgatcagc tgggccaaga accctccacc
3024414DNAArtificial SequencePrimer
244catcccttgc atgg
1424529DNAArtificial SequencePrimer 245catgatcagc tgggccaaga actgatatc
2924614DNAArtificial SequencePrimer
246gtgcctactg agct
1424729DNAArtificial SequencePrimer 247catgatcagc tgggccaaga acctaatat
2924820DNAArtificial SequencePrimer
248tgatatgttt gatatattag
2024929DNAArtificial SequencePrimer 249catgatcagc tgggccaaga agctgcttt
2925017DNAArtificial SequencePrimer
250caacggaatc ccaaaag
1725129DNAArtificial SequencePrimer 251catgatcagc tgggccaaga ggctgtcaa
2925217DNAArtificial SequencePrimer
252ctgacctatg aattgac
1725329DNAArtificial SequencePrimer 253catgatcagc tgggccaaga ctgggactt
2925415DNAArtificial SequencePrimer
254aactggccta caaag
1525528DNAArtificial SequencePrimer 255catgatcagc tgggccaaga tccacatg
2825616DNAArtificial SequencePrimer
256tgtaacagca actcca
1625729DNAArtificial SequencePrimer 257catgatcagc tgggccaaga gccaatatt
2925816DNAArtificial SequencePrimer
258tagcagcaca gaaata
1625930DNAArtificial SequencePrimer 259catgatcagc tgggccaaga ccaacaacag
3026015DNAArtificial SequencePrimer
260taggtagttt cctgt
1526130DNAArtificial SequencePrimer 261catgatcagc tgggccaaga ccaacaacat
3026217DNAArtificial SequencePrimer
262taggtagttt catgttg
1726329DNAArtificial SequencePrimer 263catgatcagc tgggccaaga gctgggtgg
2926414DNAArtificial SequencePrimer
264ttcaccacct tctc
1426528DNAArtificial SequencePrimer 265catgatcagc tgggccaaga cctatctc
2826614DNAArtificial SequencePrimer
266ggtccagagg ggag
1426729DNAArtificial SequencePrimer 267catgatcagc tgggccaaga aaccaatgt
2926815DNAArtificial SequencePrimer
268tacagtagtc tgcac
1526929DNAArtificial SequencePrimer 269catgatcagc tgggccaaga gaacaggta
2927014DNAArtificial SequencePrimer
270cccagtgttc agac
1427130DNAArtificial SequencePrimer 271catgatcagc tgggccaaga gaacagatag
3027214DNAArtificial SequencePrimer
272cccagtgttt agac
1427329DNAArtificial SequencePrimer 273catgatcagc tgggccaaga acatcgtta
2927414DNAArtificial SequencePrimer
274taacactgtc tggt
1427529DNAArtificial SequencePrimer 275catgatcagc tgggccaaga gtcatcatt
2927617DNAArtificial SequencePrimer
276taatactgcc tggtaat
1727730DNAArtificial SequencePrimer 277catgatcagc tgggccaaga ttttcccatg
3027815DNAArtificial SequencePrimer
278agaggtatag ggcat
1527929DNAArtificial SequencePrimer 279catgatcagc tgggccaaga ctagtggtc
2928017DNAArtificial SequencePrimer
280gtgaaatgtt taggacc
1728129DNAArtificial SequencePrimer 281catgatcagc tgggccaaga aggcatagg
2928215DNAArtificial SequencePrimer
282ttccctttgt catcc
1528330DNAArtificial SequencePrimer 283catgatcagc tgggccaaga cagactccgg
3028414DNAArtificial SequencePrimer
284tccttcattc cacc
1428527DNAArtificial SequencePrimer 285catgatcagc tgggccaaga ccacaca
2728618DNAArtificial SequencePrimer
286tggaatgtaa ggaagtgt
1828733DNAArtificial SequencePrimer 287catgatcagc tgggccaaga acaagctttt
tgc 3328817DNAArtificial SequencePrimer
288ataagacgag caaaaag
1728930DNAArtificial SequencePrimer 289catgatcagc tgggccaaga tcagccgctg
3029014DNAArtificial SequencePrimer
290ctgtgcgtgt gaca
1429129DNAArtificial SequencePrimer 291catgatcagc tgggccaaga aggcgaagg
2929215DNAArtificial SequencePrimer
292ttccctttgt catcc
1529329DNAArtificial SequencePrimer 293catgatcagc tgggccaaga ggccgtgac
2929416DNAArtificial SequencePrimer
294taacagtctc cagtca
1629530DNAArtificial SequencePrimer 295catgatcagc tgggccaaga ggtacaatca
3029614DNAArtificial SequencePrimer
296accatcgacc gttg
1429730DNAArtificial SequencePrimer 297catgatcagc tgggccaaga ctgcctgtct
3029814DNAArtificial SequencePrimer
298acagcaggca caga
1429929DNAArtificial SequencePrimer 299catgatcagc tgggccaaga gtctgtcaa
2930017DNAArtificial SequencePrimer
300atgacctatg aattgac
1730129DNAArtificial SequencePrimer 301catgatcagc tgggccaaga cacagttgc
2930215DNAArtificial SequencePrimer
302taatctcagc tggca
1530329DNAArtificial SequencePrimer 303catgatcagc tgggccaaga atccaatca
2930418DNAArtificial SequencePrimer
304tactgcatca ggaactga
1830529DNAArtificial SequencePrimer 305catgatcagc tgggccaaga acatggtta
2930616DNAArtificial SequencePrimer
306ttgtgcttga tctaac
1630730DNAArtificial SequencePrimer 307catgatcagc tgggccaaga aaagtgtcag
3030814DNAArtificial SequencePrimer
308ccacaccgta tctg
1430929DNAArtificial SequencePrimer 309catgatcagc tgggccaaga gaaacccag
2931015DNAArtificial SequencePrimer
310agctacattg tctgc
1531128DNAArtificial SequencePrimer 311catgatcagc tgggccaaga gagaccca
2831214DNAArtificial SequencePrimer
312agctacatct ggct
1431330DNAArtificial SequencePrimer 313catgatcagc tgggccaaga ggggtatttg
3031415DNAArtificial SequencePrimer
314tgtcagtttg tcaaa
1531528DNAArtificial SequencePrimer 315catgatcagc tgggccaaga taaacgga
2831616DNAArtificial SequencePrimer
316caagtcacta gtggtt
1631729DNAArtificial SequencePrimer 317catgatcagc tgggccaaga acaggattg
2931815DNAArtificial SequencePrimer
318agggcccccc ctcaa
1531929DNAArtificial SequencePrimer 319catgatcagc tgggccaaga atgtatgtg
2932014DNAArtificial SequencePrimer
320tggtttaccg tccc
1432130DNAArtificial SequencePrimer 321catgatcagc tgggccaaga gctttgacaa
3032217DNAArtificial SequencePrimer
322cagtgcaata gtattgt
1732330DNAArtificial SequencePrimer 323catgatcagc tgggccaaga aaagcaagta
3032415DNAArtificial SequencePrimer
324taaacgtgga tgtac
1532530DNAArtificial SequencePrimer 325catgatcagc tgggccaaga tcaccaaaac
3032615DNAArtificial SequencePrimer
326taagtgcttc catgt
1532730DNAArtificial SequencePrimer 327catgatcagc tgggccaaga agaaagcact
3032817DNAArtificial SequencePrimer
328actttaacat ggaagtg
1732930DNAArtificial SequencePrimer 329catgatcagc tgggccaaga ctactaaaac
3033015DNAArtificial SequencePrimer
330taagtgcttc catgt
1533130DNAArtificial SequencePrimer 331catgatcagc tgggccaaga acactcaaac
3033215DNAArtificial SequencePrimer
332taagtgcttc catgt
1533329DNAArtificial SequencePrimer 333catgatcagc tgggccaaga cagcaggta
2933417DNAArtificial SequencePrimer
334tttaacatgg gggtacc
1733529DNAArtificial SequencePrimer 335catgatcagc tgggccaaga ccactgaaa
2933619DNAArtificial SequencePrimer
336taagtgcttc catgtttca
1933728DNAArtificial SequencePrimer 337catgatcagc tgggccaaga ttcgccct
2833818DNAArtificial SequencePrimer
338aaaagctggg ttgagagg
1833930DNAArtificial SequencePrimer 339catgatcagc tgggccaaga agaggtcgac
3034014DNAArtificial SequencePrimer
340gcacattaca cggt
1434130DNAArtificial SequencePrimer 341catgatcagc tgggccaaga ccagcagcac
3034214DNAArtificial SequencePrimer
342ccactgcccc aggt
1434330DNAArtificial SequencePrimer 343catgatcagc tgggccaaga acaccaatgc
3034414DNAArtificial SequencePrimer
344cgcatcccct aggg
1434530DNAArtificial SequencePrimer 345catgatcagc tgggccaaga acacttactg
3034614DNAArtificial SequencePrimer
346cctagtaggt gtcc
1434730DNAArtificial SequencePrimer 347catgatcagc tgggccaaga ctggaggaag
3034814DNAArtificial SequencePrimer
348cctctgggcc cttc
1434930DNAArtificial SequencePrimer 349catgatcagc tgggccaaga acggaagggc
3035014DNAArtificial SequencePrimer
350ctggccctct ctgc
1435130DNAArtificial SequencePrimer 351catgatcagc tgggccaaga tctctgcagg
3035214DNAArtificial SequencePrimer
352gcaaagcaca cggc
1435330DNAArtificial SequencePrimer 353catgatcagc tgggccaaga ttctaggata
3035414DNAArtificial SequencePrimer
354gcccctgggc ctat
1435530DNAArtificial SequencePrimer 355catgatcagc tgggccaaga aaaggcatca
3035615DNAArtificial SequencePrimer
356tccagctcct atatg
1535730DNAArtificial SequencePrimer 357catgatcagc tgggccaaga tcaacaaaat
3035817DNAArtificial SequencePrimer
358tccagcatca gtgattt
1735929DNAArtificial SequencePrimer 359catgatcagc tgggccaaga tgagctcct
2936015DNAArtificial SequencePrimer
360tccctgtcct ccagg
1536130DNAArtificial SequencePrimer 361catgatcagc tgggccaaga ggctataaag
3036214DNAArtificial SequencePrimer
362tccgtctcag ttac
1436328DNAArtificial SequencePrimer 363catgatcagc tgggccaaga gacgggtg
2836416DNAArtificial SequencePrimer
364tctcacacag aaatcg
1636530DNAArtificial SequencePrimer 365catgatcagc tgggccaaga gccctggact
3036614DNAArtificial SequencePrimer
366tgctgactcc tagt
1436730DNAArtificial SequencePrimer 367catgatcagc tgggccaaga agaggcaggc
3036814DNAArtificial SequencePrimer
368tgtctgcccg catg
1436930DNAArtificial SequencePrimer 369catgatcagc tgggccaaga tacagatgga
3037015DNAArtificial SequencePrimer
370aattgcacgg tatcc
1537130DNAArtificial SequencePrimer 371catgatcagc tgggccaaga tcaccattgc
3037217DNAArtificial SequencePrimer
372aattgcactt tagcaat
1737330DNAArtificial SequencePrimer 373catgatcagc tgggccaaga aaacgtggaa
3037418DNAArtificial SequencePrimer
374acatagagga aattccac
1837530DNAArtificial SequencePrimer 375catgatcagc tgggccaaga ccaggttcca
3037614DNAArtificial SequencePrimer
376gcctgctggg gtgg
1437730DNAArtificial SequencePrimer 377catgatcagc tgggccaaga acactcaaaa
3037814DNAArtificial SequencePrimer
378gtgccgccat cttt
1437930DNAArtificial SequencePrimer 379catgatcagc tgggccaaga acgctcaaat
3038014DNAArtificial SequencePrimer
380aaagtgctgc gaca
1438130DNAArtificial SequencePrimer 381catgatcagc tgggccaaga ggaaagcgcc
3038214DNAArtificial SequencePrimer
382actcaaaatg gggg
1438330DNAArtificial SequencePrimer 383catgatcagc tgggccaaga acaccccaaa
3038418DNAArtificial SequencePrimer
384gaagtgcttc gattttgg
1838529DNAArtificial SequencePrimer 385catgatcagc tgggccaaga cacttatca
2938620DNAArtificial SequencePrimer
386ttataataca acctgataag
2038730DNAArtificial SequencePrimer 387catgatcagc tgggccaaga tcacgcgagc
3038814DNAArtificial SequencePrimer
388tttgttcgtt cggc
1438928DNAArtificial SequencePrimer 389catgatcagc tgggccaaga aacatgga
2839018DNAArtificial SequencePrimer
390atcatagagg aaaatcca
1839130DNAArtificial SequencePrimer 391catgatcagc tgggccaaga acacaggacc
3039214DNAArtificial SequencePrimer
392ctcctgactc cagg
1439327DNAArtificial SequencePrimer 393catgatcagc tgggccaaga tacgttc
2739417DNAArtificial SequencePrimer
394tggtagacta tggaacg
1739530DNAArtificial SequencePrimer 395catgatcagc tgggccaaga gcgcatgttc
3039614DNAArtificial SequencePrimer
396tggttgacca taga
1439730DNAArtificial SequencePrimer 397catgatcagc tgggccaaga aagatgtgga
3039818DNAArtificial SequencePrimer
398tatgtaatat ggtccaca
1839929DNAArtificial SequencePrimer 399catgatcagc tgggccaaga acagagagc
2940016DNAArtificial SequencePrimer
400tatacaaggg caagct
1640130DNAArtificial SequencePrimer 401catgatcagc tgggccaaga cgaatccacc
3040215DNAArtificial SequencePrimer
402gaagttgttc gtggt
1540330DNAArtificial SequencePrimer 403catgatcagc tgggccaaga agccacaatc
3040418DNAArtificial SequencePrimer
404agatcagaag gtgattgt
1840529DNAArtificial SequencePrimer 405catgatcagc tgggccaaga acaggccat
2940617DNAArtificial SequencePrimer
406aatataacac agatggc
1740730DNAArtificial SequencePrimer 407catgatcagc tgggccaaga acggctagtg
3040818DNAArtificial SequencePrimer
408acttcacctg gtccacta
1840930DNAArtificial SequencePrimer 409catgatcagc tgggccaaga ggccttctga
3041014DNAArtificial SequencePrimer
410ctggacttag ggtc
1441130DNAArtificial SequencePrimer 411catgatcagc tgggccaaga ggccttctga
3041214DNAArtificial SequencePrimer
412ctggacttgg agtc
1441330DNAArtificial SequencePrimer 413catgatcagc tgggccaaga ctgaggggcc
3041414DNAArtificial SequencePrimer
414agctcggtct gagg
1441530DNAArtificial SequencePrimer 415catgatcagc tgggccaaga ttcaaaacat
3041619DNAArtificial SequencePrimer
416cagcagcaat tcatgtttt
1941730DNAArtificial SequencePrimer 417catgatcagc tgggccaaga ggcggacacg
3041814DNAArtificial SequencePrimer
418atcgggaatg tcgt
1441931DNAArtificial SequencePrimer 419catgatcagc tgggccaaga acggttttac c
3142018DNAArtificial SequencePrimer
420taatactgtc tggtaaaa
1842130DNAArtificial SequencePrimer 421catgatcagc tgggccaaga tgcatgacgg
3042214DNAArtificial SequencePrimer
422tgtcttgcag gccg
1442330DNAArtificial SequencePrimer 423catgatcagc tgggccaaga atgggacatc
3042416DNAArtificial SequencePrimer
424ttgcatatgt aggatg
1642530DNAArtificial SequencePrimer 425catgatcagc tgggccaaga accagctaac
3042618DNAArtificial SequencePrimer
426tggcagtgta ttgttagc
1842730DNAArtificial SequencePrimer 427catgatcagc tgggccaaga tattaggaac
3042815DNAArtificial SequencePrimer
428tttttgcgat gtgtt
1542930DNAArtificial SequencePrimer 429catgatcagc tgggccaaga aaactcagta
3043019DNAArtificial SequencePrimer
430aaaccgttac cattactga
1943128DNAArtificial SequencePrimer 431catgatcagc tgggccaaga aactatac
2843215DNAArtificial SequencePrimer
432tgaggtagta ggttg
1543328DNAArtificial SequencePrimer 433catgatcagc tgggccaaga aaccacac
2843428DNAArtificial SequencePrimer
434catgatcagc tgggccaaga aaccatac
2843528DNAArtificial SequencePrimer 435catgatcagc tgggccaaga actatgca
2843615DNAArtificial SequencePrimer
436agaggtagta ggttg
1543728DNAArtificial SequencePrimer 437catgatcagc tgggccaaga actataca
2843815DNAArtificial SequencePrimer
438tgaggtagga ggttg
1543928DNAArtificial SequencePrimer 439catgatcagc tgggccaaga aactatac
2844015DNAArtificial SequencePrimer
440tgaggtagta gattg
1544128DNAArtificial SequencePrimer 441catgatcagc tgggccaaga actgtaca
2844215DNAArtificial SequencePrimer
442tgaggtagta gtttg
1544328DNAArtificial SequencePrimer 443catgatcagc tgggccaaga acagcaca
2844415DNAArtificial SequencePrimer
444tgaggtagta gtttg
1544530DNAArtificial SequencePrimer 445catgatcagc tgggccaaga acaaaagttg
3044616DNAArtificial SequencePrimer
446atcacacaaa ggcaac
1644727DNAArtificial SequencePrimer 447catgatcagc tgggccaaga acgtgga
2744817DNAArtificial SequencePrimer
448atcatagagg aaaatcc
1744930DNAArtificial SequencePrimer 449catgatcagc tgggccaaga acagttcttc
3045014DNAArtificial SequencePrimer
450aagctgccag ttga
1445129DNAArtificial SequencePrimer 451catgatcagc tgggccaaga ccatcatta
2945214DNAArtificial SequencePrimer
452taatactgcc gggt
1445330DNAArtificial SequencePrimer 453catgatcagc tgggccaaga ctgttcctgc
3045414DNAArtificial SequencePrimer
454tggctcagtt cagc
1445530DNAArtificial SequencePrimer 455catgatcagc tgggccaaga accgatttca
3045616DNAArtificial SequencePrimer
456tagcaccatt tgaaat
1645730DNAArtificial SequencePrimer 457catgatcagc tgggccaaga tatctgcact
3045815DNAArtificial SequencePrimer
458taaggtgcat ctagt
1545930DNAArtificial SequencePrimer 459catgatcagc tgggccaaga gaactgcctt
3046014DNAArtificial SequencePrimer
460tggagagaaa ggca
1446128DNAArtificial SequencePrimer 461catgatcagc tgggccaaga cccaccga
2846216DNAArtificial SequencePrimer
462aacattcatt gctgtc
1646330DNAArtificial SequencePrimer 463catgatcagc tgggccaaga acaagtgccc
3046414DNAArtificial SequencePrimer
464actgcagtga gggc
1446530DNAArtificial SequencePrimer 465catgatcagc tgggccaaga agccacagtc
3046618DNAArtificial SequencePrimer
466agatcagaag gtgactgt
1846728DNAArtificial SequencePrimer 467catgatcagc tgggccaaga acgtggat
2846819DNAArtificial SequencePrimer
468atcgtagagg aaaatccac
1946917DNAArtificial SequencePrimer 469atgacctatg atttgac
1747029DNAArtificial SequencePrimer
470catgatcagc tgggccaaga tgtgaacaa
2947117DNAArtificial SequencePrimer 471attcctagaa attgttc
1747229DNAArtificial SequencePrimer
472catgatcagc tgggccaaga tacctgcac
2947316DNAArtificial SequencePrimer 473caaagtgcta acagtg
1647430DNAArtificial SequencePrimer
474catgatcagc tgggccaaga tccaaaacat
3047530DNAArtificial SequencePrimer 475catgatcagc tgggccaaga gaacaggtag
3047614DNAArtificial SequencePrimer
476cctagtaggt gctc
1447715DNAArtificial SequencePrimer 477actagactga ggctc
1547814DNAArtificial SequencePrimer
478gcaaagcaca gggc
1447931DNAArtificial SequencePrimer 479catgatcagc tgggccaaga acggcattac c
3148018DNAArtificial SequencePrimer
480taatactgtc tggtaatg
1848129DNAArtificial SequencePrimer 481catgatcagc tgggccaaga atccagtca
2948214DNAArtificial SequencePrimer
482taggcagtgt aatt
1448318DNAArtificial SequencePrimer 483tatgtagtat ggtccaca
1848430DNAArtificial SequencePrimer
484catgatcagc tgggccaaga gcactggact
3048514DNAArtificial SequencePrimer 485tgctgacccc tagt
1448629DNAArtificial SequencePrimer
486catgatcagc tgggccaaga aacaaaatc
2948718DNAArtificial SequencePrimer 487tggaagactt gtgatttt
1848814DNAArtificial SequencePrimer
488tgtctgcccg agtg
1448920DNAArtificial SequencePrimer 489ttaatgctaa ttgtgatagg
2049015DNAArtificial SequencePrimer
490ttcagctcct atatg
1549129DNAArtificial SequencePrimer 491catgatcagc tgggccaaga aggcaaagg
2949231DNAArtificial SequencePrimer
492catgatcagc tgggccaaga acacaaattc g
3149316DNAArtificial SequencePrimer 493ccctgtagaa ccgaat
1649415DNAArtificial SequencePrimer
494tcacagtgaa ccggt
1549515DNAArtificial SequencePrimer 495agctggtgtt gtgaa
1549616DNAArtificial SequencePrimer
496tgagatgaag cactgt
1649716DNAArtificial SequencePrimer 497tctcccaacc cttgta
1649815DNAArtificial SequencePrimer
498aacattcaac gctgt
1549916DNAArtificial SequencePrimer 499tgtaacagca actcca
1650030DNAArtificial SequenceSynthetic
500catgatcagc tgggccaaga aagaagtgca
3050129DNAArtificial SequenceSynthetic 501catgatcagc tgggccaaga aagaagtgc
2950228DNAArtificial
SequenceSynthetic 502catgatcagc tgggccaaga aagaagtg
2850327DNAArtificial SequenceSynthetic 503catgatcagc
tgggccaaga aagaagt
2750417DNAArtificial SequenceSynthetic 504aaacaaacat ggtgcac
1750515DNAArtificial
SequenceSynthetic 505aaacaaacat ggtgc
1550630DNAArtificial SequenceSynthetic 506catgatcagc
tgggccaaga ggcacacaaa
3050729DNAArtificial SequenceSynthetic 507catgatcagc tgggccaaga ggcacacaa
2950828DNAArtificial
SequenceSynthetic 508catgatcagc tgggccaaga ggcacaca
2850927DNAArtificial SequenceSynthetic 509catgatcagc
tgggccaaga ggcacac
2751018DNAArtificial SequenceSynthetic 510aaagtgcttc cactttgt
1851116DNAArtificial
SequenceSynthetic 511aaagtgcttc cacttt
1651230DNAArtificial SequenceSynthetic 512catgatcagc
tgggccaaga gacaaacaaa
3051329DNAArtificial SequenceSynthetic 513catgatcagc tgggccaaga gacaaacaa
2951428DNAArtificial
SequenceSynthetic 514catgatcagc tgggccaaga gacaaaca
2851527DNAArtificial SequenceSynthetic 515catgatcagc
tgggccaaga gacaaac
2751618DNAArtificial SequenceSynthetic 516aaagtgcatc cattttgt
1851716DNAArtificial
SequenceSynthetic 517aaagtgcatc catttt
1651830DNAArtificial SequenceSynthetic 518catgatcagc
tgggccaaga gtaacactct
3051929DNAArtificial SequenceSynthetic 519catgatcagc tgggccaaga gtaacactc
2952028DNAArtificial
SequenceSynthetic 520catgatcagc tgggccaaga gtaacact
2852127DNAArtificial SequenceSynthetic 521catgatcagc
tgggccaaga gtaacac
2752220DNAArtificial SequenceSynthetic 522aaagtgcatc cttttagagt
2052318DNAArtificial
SequenceSynthetic 523aaagtgcatc cttttaga
1852430DNAArtificial SequenceSynthetic 524catgatcagc
tgggccaaga aaacctctaa
3052529DNAArtificial SequenceSynthetic 525catgatcagc tgggccaaga aaacctcta
2952628DNAArtificial
SequenceSynthetic 526catgatcagc tgggccaaga aaacctct
2852727DNAArtificial SequenceSynthetic 527catgatcagc
tgggccaaga aaacctc
2752817DNAArtificial SequenceSynthetic 528aaagtgcatc cttttag
1752915DNAArtificial
SequenceSynthetic 529aaagtgcatc ctttt
1553030DNAArtificial SequenceSynthetic 530catgatcagc
tgggccaaga atcctctaaa
3053129DNAArtificial SequenceSynthetic 531catgatcagc tgggccaaga atcctctaa
2953228DNAArtificial
SequenceSynthetic 532catgatcagc tgggccaaga atcctcta
2853327DNAArtificial SequenceSynthetic 533catgatcagc
tgggccaaga atcctct
2753418DNAArtificial SequenceSynthetic 534aaagtgcatc tttttaga
1853516DNAArtificial
SequenceSynthetic 535aaagtgcatc ttttta
1653630DNAArtificial SequenceSynthetic 536catgatcagc
tgggccaaga acactctaaa
3053729DNAArtificial SequenceSynthetic 537catgatcagc tgggccaaga acactctaa
2953828DNAArtificial
SequenceSynthetic 538catgatcagc tgggccaaga acactcta
2853927DNAArtificial SequenceSynthetic 539catgatcagc
tgggccaaga acactct
2754018DNAArtificial SequenceSynthetic 540caaagtgcct ccctttag
1854116DNAArtificial
SequenceSynthetic 541caaagtgcct cccttt
1654230DNAArtificial SequenceSynthetic 542catgatcagc
tgggccaaga acagtccaaa
3054329DNAArtificial SequenceSynthetic 543catgatcagc tgggccaaga acagtccaa
2954428DNAArtificial
SequenceSynthetic 544catgatcagc tgggccaaga acagtcca
2854527DNAArtificial SequenceSynthetic 545catgatcagc
tgggccaaga acagtcc
2754617DNAArtificial SequenceSynthetic 546aaagtgcttc cctttgg
1754715DNAArtificial
SequenceSynthetic 547aaagtgcttc ccttt
1554830DNAArtificial SequenceSynthetic 548catgatcagc
tgggccaaga ccctctaaaa
3054929DNAArtificial SequenceSynthetic 549catgatcagc tgggccaaga ccctctaaa
2955028DNAArtificial
SequenceSynthetic 550catgatcagc tgggccaaga ccctctaa
2855127DNAArtificial SequenceSynthetic 551catgatcagc
tgggccaaga ccctcta
2755217DNAArtificial SequenceSynthetic 552aaagtgcttc cttttag
1755316DNAArtificial
SequenceSynthetic 553aaagtgcttc ctttta
1655430DNAArtificial SequenceSynthetic 554catgatcagc
tgggccaaga aacccaccaa
3055529DNAArtificial SequenceSynthetic 555catgatcagc tgggccaaga aacccacca
2955628DNAArtificial
SequenceSynthetic 556catgatcagc tgggccaaga aacccacc
2855727DNAArtificial SequenceSynthetic 557catgatcagc
tgggccaaga aacccac
2755818DNAArtificial SequenceSynthetic 558aaagtgcttc tctttggt
1855916DNAArtificial
SequenceSynthetic 559aaagtgcttc tctttg
1656030DNAArtificial SequenceSynthetic 560catgatcagc
tgggccaaga ccctcaaaaa
3056129DNAArtificial SequenceSynthetic 561catgatcagc tgggccaaga ccctcaaaa
2956228DNAArtificial
SequenceSynthetic 562catgatcagc tgggccaaga ccctcaaa
2856327DNAArtificial SequenceSynthetic 563catgatcagc
tgggccaaga ccctcaa
2756417DNAArtificial SequenceSynthetic 564aaagtgcttc ctttttg
1756516DNAArtificial
SequenceSynthetic 565aaagtgcttc cttttt
1656630DNAArtificial SequenceSynthetic 566catgatcagc
tgggccaaga aaccctctaa
3056729DNAArtificial SequenceSynthetic 567catgatcagc tgggccaaga aaccctcta
2956828DNAArtificial
SequenceSynthetic 568catgatcagc tgggccaaga aaccctct
2856927DNAArtificial SequenceSynthetic 569catgatcagc
tgggccaaga aaccctc
2757017DNAArtificial SequenceSynthetic 570aagtgcttcc ttttaga
1757115DNAArtificial
SequenceSynthetic 571aagtgcttcc tttta
1557230DNAArtificial SequenceSynthetic 572catgatcagc
tgggccaaga aaaaaggtta
3057329DNAArtificial SequenceSynthetic 573catgatcagc tgggccaaga aaaaaggtt
2957428DNAArtificial
SequenceSynthetic 574catgatcagc tgggccaaga aaaaaggt
2857527DNAArtificial SequenceSynthetic 575catgatcagc
tgggccaaga aaaaagg
2757617DNAArtificial SequenceSynthetic 576aacacaccca gctaacc
1757715DNAArtificial
SequenceSynthetic 577aacacaccca gctaa
1557830DNAArtificial SequenceSynthetic 578catgatcagc
tgggccaaga aacccaccga
3057929DNAArtificial SequenceSynthetic 579catgatcagc tgggccaaga aacccaccg
2958028DNAArtificial
SequenceSynthetic 580catgatcagc tgggccaaga aacccacc
2858127DNAArtificial SequenceSynthetic 581catgatcagc
tgggccaaga aacccac
2758219DNAArtificial SequenceSynthetic 582aacattcatt gttgtcggt
1958317DNAArtificial
SequenceSynthetic 583aacattcatt gttgtcg
1758430DNAArtificial SequenceSynthetic 584catgatcagc
tgggccaaga aaagcgggac
3058529DNAArtificial SequenceSynthetic 585catgatcagc tgggccaaga aaagcggga
2958628DNAArtificial
SequenceSynthetic 586catgatcagc tgggccaaga aaagcggg
2858727DNAArtificial SequenceSynthetic 587catgatcagc
tgggccaaga aaagcgg
2758819DNAArtificial SequenceSynthetic 588aactggccct caaagtccc
1958917DNAArtificial
SequenceSynthetic 589aactggccct caaagtc
1759030DNAArtificial SequenceSynthetic 590catgatcagc
tgggccaaga actcacacct
3059129DNAArtificial SequenceSynthetic 591catgatcagc tgggccaaga actcacacc
2959228DNAArtificial
SequenceSynthetic 592catgatcagc tgggccaaga actcacac
2859327DNAArtificial SequenceSynthetic 593catgatcagc
tgggccaaga actcaca
2759419DNAArtificial SequenceSynthetic 594aatccttgga acctaggtg
1959517DNAArtificial
SequenceSynthetic 595aatccttgga acctagg
1759630DNAArtificial SequenceSynthetic 596catgatcagc
tgggccaaga ttcacaccta
3059729DNAArtificial SequenceSynthetic 597catgatcagc tgggccaaga ttcacacct
2959828DNAArtificial
SequenceSynthetic 598catgatcagc tgggccaaga ttcacacc
2859927DNAArtificial SequenceSynthetic 599catgatcagc
tgggccaaga ttcacac
2760018DNAArtificial SequenceSynthetic 600aatccttgga acctaggt
1860116DNAArtificial
SequenceSynthetic 601aatccttgga acctag
1660230DNAArtificial SequenceSynthetic 602catgatcagc
tgggccaaga cagaatcctt
3060329DNAArtificial SequenceSynthetic 603catgatcagc tgggccaaga cagaatcct
2960428DNAArtificial
SequenceSynthetic 604catgatcagc tgggccaaga cagaatcc
2860527DNAArtificial SequenceSynthetic 605catgatcagc
tgggccaaga cagaatc
2760617DNAArtificial SequenceSynthetic 606atgcacctgg gcaagga
1760715DNAArtificial
SequenceSynthetic 607atgcacctgg gcaag
1560830DNAArtificial SequenceSynthetic 608catgatcagc
tgggccaaga ctgaaccctt
3060929DNAArtificial SequenceSynthetic 609catgatcagc tgggccaaga ctgaaccct
2961028DNAArtificial
SequenceSynthetic 610catgatcagc tgggccaaga ctgaaccc
2861127DNAArtificial SequenceSynthetic 611catgatcagc
tgggccaaga ctgaacc
2761217DNAArtificial SequenceSynthetic 612atgcacctgg gcaaggg
1761315DNAArtificial
SequenceSynthetic 613atgcacctgg gcaag
1561430DNAArtificial SequenceSynthetic 614catgatcagc
tgggccaaga tctcacccag
3061529DNAArtificial SequenceSynthetic 615catgatcagc tgggccaaga tctcaccca
2961628DNAArtificial
SequenceSynthetic 616catgatcagc tgggccaaga tctcaccc
2861727DNAArtificial SequenceSynthetic 617catgatcagc
tgggccaaga tctcacc
2761817DNAArtificial SequenceSynthetic 618aatcctttgt ccctggg
1761916DNAArtificial
SequenceSynthetic 619aatcctttgt ccctgg
1662030DNAArtificial SequenceSynthetic 620catgatcagc
tgggccaaga tttcacccag
3062129DNAArtificial SequenceSynthetic 621catgatcagc tgggccaaga tttcaccca
2962228DNAArtificial
SequenceSynthetic 622catgatcagc tgggccaaga tttcaccc
2862327DNAArtificial SequenceSynthetic 623catgatcagc
tgggccaaga tttcacc
2762417DNAArtificial SequenceSynthetic 624aatcctttgt ccctggg
1762515DNAArtificial
SequenceSynthetic 625aatcctttgt ccctg
1562630DNAArtificial SequenceSynthetic 626catgatcagc
tgggccaaga agtggatgac
3062729DNAArtificial SequenceSynthetic 627catgatcagc tgggccaaga agtggatga
2962828DNAArtificial
SequenceSynthetic 628catgatcagc tgggccaaga agtggatg
2862927DNAArtificial SequenceSynthetic 629catgatcagc
tgggccaaga agtggat
2763016DNAArtificial SequenceSynthetic 630aatcgtacag ggtcat
1663114DNAArtificial
SequenceSynthetic 631aatcgtacag ggtc
1463230DNAArtificial SequenceSynthetic 632catgatcagc
tgggccaaga gctgccgtat
3063329DNAArtificial SequenceSynthetic 633catgatcagc tgggccaaga gctgccgta
2963428DNAArtificial
SequenceSynthetic 634catgatcagc tgggccaaga gctgccgt
2863527DNAArtificial SequenceSynthetic 635catgatcagc
tgggccaaga gctgccg
2763618DNAArtificial SequenceSynthetic 636agtgacatca catatacg
1863717DNAArtificial
SequenceSynthetic 637agtgacatca catatac
1763830DNAArtificial SequenceSynthetic 638catgatcagc
tgggccaaga gctgccatat
3063929DNAArtificial SequenceSynthetic 639catgatcagc tgggccaaga gctgccata
2964028DNAArtificial
SequenceSynthetic 640catgatcagc tgggccaaga gctgccat
2864127DNAArtificial SequenceSynthetic 641catgatcagc
tgggccaaga gctgcca
2764218DNAArtificial SequenceSynthetic 642aatgacacca catatatg
1864315DNAArtificial
SequenceSynthetic 643aatgacacca catat
1564430DNAArtificial SequenceSynthetic 644catgatcagc
tgggccaaga gctgccatat
3064529DNAArtificial SequenceSynthetic 645catgatcagc tgggccaaga gctgccata
2964628DNAArtificial
SequenceSynthetic 646catgatcagc tgggccaaga gctgccat
2864727DNAArtificial SequenceSynthetic 647catgatcagc
tgggccaaga gctgcca
2764818DNAArtificial SequenceSynthetic 648aatgacatca catatatg
1864917DNAArtificial
SequenceSynthetic 649aatgacatca catatat
1765030DNAArtificial SequenceSynthetic 650catgatcagc
tgggccaaga tcaacgggag
3065129DNAArtificial SequenceSynthetic 651catgatcagc tgggccaaga tcaacggga
2965228DNAArtificial
SequenceSynthetic 652catgatcagc tgggccaaga tcaacggg
2865327DNAArtificial SequenceSynthetic 653catgatcagc
tgggccaaga tcaacgg
2765418DNAArtificial SequenceSynthetic 654aatgacacga tcactccc
1865516DNAArtificial
SequenceSynthetic 655aatgacacga tcactc
1665630DNAArtificial SequenceSynthetic 656catgatcagc
tgggccaaga tgcacaaccc
3065729DNAArtificial SequenceSynthetic 657catgatcagc tgggccaaga tgcacaacc
2965828DNAArtificial
SequenceSynthetic 658catgatcagc tgggccaaga tgcacaac
2865927DNAArtificial SequenceSynthetic 659catgatcagc
tgggccaaga tgcacaa
2766018DNAArtificial SequenceSynthetic 660aatggcgcca ctagggtt
1866116DNAArtificial
SequenceSynthetic 661aatggcgcca ctaggg
1666230DNAArtificial SequenceSynthetic 662catgatcagc
tgggccaaga gaattcatca
3066329DNAArtificial SequenceSynthetic 663catgatcagc tgggccaaga gaattcatc
2966428DNAArtificial
SequenceSynthetic 664catgatcagc tgggccaaga gaattcat
2866527DNAArtificial SequenceSynthetic 665catgatcagc
tgggccaaga gaattca
2766617DNAArtificial SequenceSynthetic 666agaggctggc cgtgatg
1766715DNAArtificial
SequenceSynthetic 667agaggctggc cgtga
1566830DNAArtificial SequenceSynthetic 668catgatcagc
tgggccaaga agagaggaga
3066929DNAArtificial SequenceSynthetic 669catgatcagc tgggccaaga agagaggag
2967028DNAArtificial
SequenceSynthetic 670catgatcagc tgggccaaga agagagga
2867127DNAArtificial SequenceSynthetic 671catgatcagc
tgggccaaga agagagg
2767218DNAArtificial SequenceSynthetic 672agtcatacac ggctctcc
1867316DNAArtificial
SequenceSynthetic 673agtcatacac ggctct
1667430DNAArtificial SequenceSynthetic 674catgatcagc
tgggccaaga cgaatataac
3067529DNAArtificial SequenceSynthetic 675catgatcagc tgggccaaga cgaatataa
2967628DNAArtificial
SequenceSynthetic 676catgatcagc tgggccaaga cgaatata
2867727DNAArtificial SequenceSynthetic 677catgatcagc
tgggccaaga cgaatat
2767816DNAArtificial SequenceSynthetic 678agatcgaccg tgttat
1667914DNAArtificial
SequenceSynthetic 679agatcgaccg tgtt
1468030DNAArtificial SequenceSynthetic 680catgatcagc
tgggccaaga cctccagccc
3068129DNAArtificial SequenceSynthetic 681catgatcagc tgggccaaga cctccagcc
2968228DNAArtificial
SequenceSynthetic 682catgatcagc tgggccaaga cctccagc
2868327DNAArtificial SequenceSynthetic 683catgatcagc
tgggccaaga cctccag
2768419DNAArtificial SequenceSynthetic 684aggaagccct ggaggggct
1968517DNAArtificial
SequenceSynthetic 685aggaagccct ggagggg
1768630DNAArtificial SequenceSynthetic 686catgatcagc
tgggccaaga gccagctaac
3068729DNAArtificial SequenceSynthetic 687catgatcagc tgggccaaga gccagctaa
2968828DNAArtificial
SequenceSynthetic 688catgatcagc tgggccaaga gccagcta
2868927DNAArtificial SequenceSynthetic 689catgatcagc
tgggccaaga gccagct
2769017DNAArtificial SequenceSynthetic 690aggcagtgta ttgttag
1769115DNAArtificial
SequenceSynthetic 691aggcagtgta ttgtt
1569230DNAArtificial SequenceSynthetic 692catgatcagc
tgggccaaga ccagctagca
3069329DNAArtificial SequenceSynthetic 693catgatcagc tgggccaaga ccagctagc
2969428DNAArtificial
SequenceSynthetic 694catgatcagc tgggccaaga ccagctag
2869527DNAArtificial SequenceSynthetic 695catgatcagc
tgggccaaga ccagcta
2769616DNAArtificial SequenceSynthetic 696aggcagtgca ttgcta
1669714DNAArtificial
SequenceSynthetic 697aggcagtgca ttgc
1469830DNAArtificial SequenceSynthetic 698catgatcagc
tgggccaaga cgagccaggt
3069929DNAArtificial SequenceSynthetic 699catgatcagc tgggccaaga cgagccagg
2970028DNAArtificial
SequenceSynthetic 700catgatcagc tgggccaaga cgagccag
2870127DNAArtificial SequenceSynthetic 701catgatcagc
tgggccaaga cgagcca
2770215DNAArtificial SequenceSynthetic 702aggcagtgcg acctg
1570313DNAArtificial
SequenceSynthetic 703aggcagtgcg acc
1370430DNAArtificial SequenceSynthetic 704catgatcagc
tgggccaaga caaagttgct
3070529DNAArtificial SequenceSynthetic 705catgatcagc tgggccaaga caaagttgc
2970628DNAArtificial
SequenceSynthetic 706catgatcagc tgggccaaga caaagttg
2870727DNAArtificial SequenceSynthetic 707catgatcagc
tgggccaaga caaagtt
2770817DNAArtificial SequenceSynthetic 708aggttacccg agcaact
1770915DNAArtificial
SequenceSynthetic 709aggttacccg agcaa
1571030DNAArtificial SequenceSynthetic 710catgatcagc
tgggccaaga aaggggttca
3071129DNAArtificial SequenceSynthetic 711catgatcagc tgggccaaga aaggggttc
2971228DNAArtificial
SequenceSynthetic 712catgatcagc tgggccaaga aaggggtt
2871327DNAArtificial SequenceSynthetic 713catgatcagc
tgggccaaga aaggggt
2771418DNAArtificial SequenceSynthetic 714gaatgttgct cggtgaac
1871516DNAArtificial
SequenceSynthetic 715gaatgttgct cggtga
1671630DNAArtificial SequenceSynthetic 716catgatcagc
tgggccaaga cctcatggaa
3071729DNAArtificial SequenceSynthetic 717catgatcagc tgggccaaga cctcatgga
2971828DNAArtificial
SequenceSynthetic 718catgatcagc tgggccaaga cctcatgg
2871927DNAArtificial SequenceSynthetic 719catgatcagc
tgggccaaga cctcatg
2772017DNAArtificial SequenceSynthetic 720agtggggaac ccttcca
1772115DNAArtificial
SequenceSynthetic 721agtggggaac ccttc
1572230DNAArtificial SequenceSynthetic 722catgatcagc
tgggccaaga tatgaacaat
3072329DNAArtificial SequenceSynthetic 723catgatcagc tgggccaaga tatgaacaa
2972428DNAArtificial
SequenceSynthetic 724catgatcagc tgggccaaga tatgaaca
2872527DNAArtificial SequenceSynthetic 725catgatcagc
tgggccaaga tatgaac
2772617DNAArtificial SequenceSynthetic 726attcctagaa attgttc
1772715DNAArtificial
SequenceSynthetic 727attcctagaa attgt
1572830DNAArtificial SequenceSynthetic 728catgatcagc
tgggccaaga tgtgaacaat
3072929DNAArtificial SequenceSynthetic 729catgatcagc tgggccaaga tgtgaacaa
2973028DNAArtificial
SequenceSynthetic 730catgatcagc tgggccaaga tgtgaaca
2873127DNAArtificial SequenceSynthetic 731catgatcagc
tgggccaaga tgtgaac
2773216DNAArtificial SequenceSynthetic 732attcctagaa attgtt
1673316DNAArtificial
SequenceSynthetic 733attcctagaa attgtt
1673430DNAArtificial SequenceSynthetic 734catgatcagc
tgggccaaga acctgcacta
3073529DNAArtificial SequenceSynthetic 735catgatcagc tgggccaaga acctgcact
2973628DNAArtificial
SequenceSynthetic 736catgatcagc tgggccaaga acctgcac
2873727DNAArtificial SequenceSynthetic 737catgatcagc
tgggccaaga acctgca
2773818DNAArtificial SequenceSynthetic 738caaagtgctc atagtgca
1873916DNAArtificial
SequenceSynthetic 739caaagtgctc atagtg
1674030DNAArtificial SequenceSynthetic 740catgatcagc
tgggccaaga cagcatggag
3074129DNAArtificial SequenceSynthetic 741catgatcagc tgggccaaga cagcatgga
2974228DNAArtificial
SequenceSynthetic 742catgatcagc tgggccaaga cagcatgg
2874327DNAArtificial SequenceSynthetic 743catgatcagc
tgggccaaga cagcatg
2774417DNAArtificial SequenceSynthetic 744caacctggag gactcca
1774515DNAArtificial
SequenceSynthetic 745caacctggag gactc
1574630DNAArtificial SequenceSynthetic 746catgatcagc
tgggccaaga acaaaccaca
3074729DNAArtificial SequenceSynthetic 747catgatcagc tgggccaaga acaaaccac
2974828DNAArtificial
SequenceSynthetic 748catgatcagc tgggccaaga acaaacca
2874927DNAArtificial SequenceSynthetic 749catgatcagc
tgggccaaga acaaacc
2775016DNAArtificial SequenceSynthetic 750cagcagcaca ctgtgg
1675115DNAArtificial
SequenceSynthetic 751cagcagcaca ctgtg
1575230DNAArtificial SequenceSynthetic 752catgatcagc
tgggccaaga tgctttgaca
3075329DNAArtificial SequenceSynthetic 753catgatcagc tgggccaaga tgctttgac
2975428DNAArtificial
SequenceSynthetic 754catgatcagc tgggccaaga tgctttga
2875527DNAArtificial SequenceSynthetic 755catgatcagc
tgggccaaga tgctttg
2775619DNAArtificial SequenceSynthetic 756cagtgcaatg atattgtca
1975717DNAArtificial
SequenceSynthetic 757cagtgcaatg atattgt
1775830DNAArtificial SequenceSynthetic 758catgatcagc
tgggccaaga tgctttgaca
3075929DNAArtificial SequenceSynthetic 759catgatcagc tgggccaaga tgctttgac
2976028DNAArtificial
SequenceSynthetic 760catgatcagc tgggccaaga tgctttga
2876127DNAArtificial SequenceSynthetic 761catgatcagc
tgggccaaga tgctttg
2776219DNAArtificial SequenceSynthetic 762cagtgcaatg gtattgtca
1976317DNAArtificial
SequenceSynthetic 763cagtgcaatg gtattgt
1776430DNAArtificial SequenceSynthetic 764catgatcagc
tgggccaaga ttcccccttt
3076529DNAArtificial SequenceSynthetic 765catgatcagc tgggccaaga ttccccctt
2976628DNAArtificial
SequenceSynthetic 766catgatcagc tgggccaaga ttccccct
2876727DNAArtificial SequenceSynthetic 767catgatcagc
tgggccaaga ttccccc
2776817DNAArtificial SequenceSynthetic 768cagtgcaatt aaaaggg
1776915DNAArtificial
SequenceSynthetic 769cagtgcaatt aaaag
1577030DNAArtificial SequenceSynthetic 770catgatcagc
tgggccaaga acggtcctac
3077129DNAArtificial SequenceSynthetic 771catgatcagc tgggccaaga acggtccta
2977228DNAArtificial
SequenceSynthetic 772catgatcagc tgggccaaga acggtcct
2877327DNAArtificial SequenceSynthetic 773catgatcagc
tgggccaaga acggtcc
2777417DNAArtificial SequenceSynthetic 774catgccttga gtgtagg
1777515DNAArtificial
SequenceSynthetic 775catgccttga gtgta
1577630DNAArtificial SequenceSynthetic 776catgatcagc
tgggccaaga ttgagagtgc
3077729DNAArtificial SequenceSynthetic 777catgatcagc tgggccaaga ttgagagtg
2977828DNAArtificial
SequenceSynthetic 778catgatcagc tgggccaaga ttgagagt
2877927DNAArtificial SequenceSynthetic 779catgatcagc
tgggccaaga ttgagag
2778016DNAArtificial SequenceSynthetic 780cccagataat ggcact
1678114DNAArtificial
SequenceSynthetic 781cccagataat ggca
1478230DNAArtificial SequenceSynthetic 782catgatcagc
tgggccaaga ttgagagtgc
3078329DNAArtificial SequenceSynthetic 783catgatcagc tgggccaaga ttgagagtg
2978428DNAArtificial
SequenceSynthetic 784catgatcagc tgggccaaga ttgagagt
2878527DNAArtificial SequenceSynthetic 785catgatcagc
tgggccaaga ttgagag
2778616DNAArtificial SequenceSynthetic 786cccagataat agcact
1678714DNAArtificial
SequenceSynthetic 787cccagataat agca
1478830DNAArtificial SequenceSynthetic 788catgatcagc
tgggccaaga acacaccaag
3078929DNAArtificial SequenceSynthetic 789catgatcagc tgggccaaga acacaccaa
2979028DNAArtificial
SequenceSynthetic 790catgatcagc tgggccaaga acacacca
2879127DNAArtificial SequenceSynthetic 791catgatcagc
tgggccaaga acacacc
2779218DNAArtificial SequenceSynthetic 792ggagaaatta tccttggt
1879317DNAArtificial
SequenceSynthetic 793ggagaaatta tccttgg
1779430DNAArtificial SequenceSynthetic 794catgatcagc
tgggccaaga gaggaaacca
3079529DNAArtificial SequenceSynthetic 795catgatcagc tgggccaaga gaggaaacc
2979628DNAArtificial
SequenceSynthetic 796catgatcagc tgggccaaga gaggaaac
2879727DNAArtificial SequenceSynthetic 797catgatcagc
tgggccaaga gaggaaa
2779817DNAArtificial SequenceSynthetic 798gtcaacactt gctggtt
1779915DNAArtificial
SequenceSynthetic 799gtcaacactt gctgg
1580030DNAArtificial SequenceSynthetic 800catgatcagc
tgggccaaga ggaaaccagc
3080129DNAArtificial SequenceSynthetic 801catgatcagc tgggccaaga ggaaaccag
2980228DNAArtificial
SequenceSynthetic 802catgatcagc tgggccaaga ggaaacca
2880327DNAArtificial SequenceSynthetic 803catgatcagc
tgggccaaga ggaaacc
2780417DNAArtificial SequenceSynthetic 804cgtcaacact tgctggt
1780515DNAArtificial
SequenceSynthetic 805cgtcaacact tgctg
1580630DNAArtificial SequenceSynthetic 806catgatcagc
tgggccaaga taactgcact
3080729DNAArtificial SequenceSynthetic 807catgatcagc tgggccaaga taactgcac
2980828DNAArtificial
SequenceSynthetic 808catgatcagc tgggccaaga taactgca
2880927DNAArtificial SequenceSynthetic 809catgatcagc
tgggccaaga taactgc
2781017DNAArtificial SequenceSynthetic 810taaggtgcat ctagtgc
1781115DNAArtificial
SequenceSynthetic 811taaggtgcat ctagt
1581230DNAArtificial SequenceSynthetic 812catgatcagc
tgggccaaga taacagcact
3081329DNAArtificial SequenceSynthetic 813catgatcagc tgggccaaga taacagcac
2981428DNAArtificial
SequenceSynthetic 814catgatcagc tgggccaaga taacagca
2881527DNAArtificial SequenceSynthetic 815catgatcagc
tgggccaaga taacagc
2781617DNAArtificial SequenceSynthetic 816taaggtgcat ctagtgc
1781715DNAArtificial
SequenceSynthetic 817taaggtgcat ctagt
1581830DNAArtificial SequenceSynthetic 818catgatcagc
tgggccaaga cagtactgtt
3081929DNAArtificial SequenceSynthetic 819catgatcagc tgggccaaga cagtactgt
2982028DNAArtificial
SequenceSynthetic 820catgatcagc tgggccaaga cagtactg
2882127DNAArtificial SequenceSynthetic 821catgatcagc
tgggccaaga cagtact
2782216DNAArtificial SequenceSynthetic 822tagcagcggg aacagt
1682314DNAArtificial
SequenceSynthetic 823tagcagcggg aaca
1482430DNAArtificial SequenceSynthetic 824catgatcagc
tgggccaaga cgatgtagtc
3082529DNAArtificial SequenceSynthetic 825catgatcagc tgggccaaga cgatgtagt
2982628DNAArtificial
SequenceSynthetic 826catgatcagc tgggccaaga cgatgtag
2882727DNAArtificial SequenceSynthetic 827catgatcagc
tgggccaaga cgatgta
2782817DNAArtificial SequenceSynthetic 828tatgtgcctt tggacta
1782915DNAArtificial
SequenceSynthetic 829tatgtgcctt tggac
1583030DNAArtificial SequenceSynthetic 830catgatcagc
tgggccaaga gaggccggga
3083129DNAArtificial SequenceSynthetic 831catgatcagc tgggccaaga gaggccggg
2983228DNAArtificial
SequenceSynthetic 832catgatcagc tgggccaaga gaggccgg
2883327DNAArtificial SequenceSynthetic 833catgatcagc
tgggccaaga gaggccg
2783416DNAArtificial SequenceSynthetic 834tattgcactc gtcccg
1683515DNAArtificial
SequenceSynthetic 835tattgcactc gtccc
1583630DNAArtificial SequenceSynthetic 836catgatcagc
tgggccaaga agaagacggg
3083729DNAArtificial SequenceSynthetic 837catgatcagc tgggccaaga agaagacgg
2983828DNAArtificial
SequenceSynthetic 838catgatcagc tgggccaaga agaagacg
2883927DNAArtificial SequenceSynthetic 839catgatcagc
tgggccaaga agaagac
2784018DNAArtificial SequenceSynthetic 840tcactcctct cctcccgt
1884116DNAArtificial
SequenceSynthetic 841tcactcctct cctccc
1684230DNAArtificial SequenceSynthetic 842catgatcagc
tgggccaaga acaagacggg
3084329DNAArtificial SequenceSynthetic 843catgatcagc tgggccaaga acaagacgg
2984428DNAArtificial
SequenceSynthetic 844catgatcagc tgggccaaga acaagacg
2884527DNAArtificial SequenceSynthetic 845catgatcagc
tgggccaaga acaagac
2784618DNAArtificial SequenceSynthetic 846tcactcctcc cctcccgt
1884716DNAArtificial
SequenceSynthetic 847tcactcctcc cctccc
1684830DNAArtificial SequenceSynthetic 848catgatcagc
tgggccaaga atcgggaggg
3084929DNAArtificial SequenceSynthetic 849catgatcagc tgggccaaga atcgggagg
2985028DNAArtificial
SequenceSynthetic 850catgatcagc tgggccaaga atcgggag
2885127DNAArtificial SequenceSynthetic 851catgatcagc
tgggccaaga atcggga
2785217DNAArtificial SequenceSynthetic 852tcaggctcag tcccctc
1785315DNAArtificial
SequenceSynthetic 853tcaggctcag tcccc
1585430DNAArtificial SequenceSynthetic 854catgatcagc
tgggccaaga caggctcaaa
3085529DNAArtificial SequenceSynthetic 855catgatcagc tgggccaaga caggctcaa
2985628DNAArtificial
SequenceSynthetic 856catgatcagc tgggccaaga caggctca
2885727DNAArtificial SequenceSynthetic 857catgatcagc
tgggccaaga caggctc
2785819DNAArtificial SequenceSynthetic 858tccctgagga gccctttga
1985917DNAArtificial
SequenceSynthetic 859tccctgagga gcccttt
1786030DNAArtificial SequenceSynthetic 860catgatcagc
tgggccaaga agagggagac
3086129DNAArtificial SequenceSynthetic 861catgatcagc tgggccaaga agagggaga
2986228DNAArtificial
SequenceSynthetic 862catgatcagc tgggccaaga agagggag
2886327DNAArtificial SequenceSynthetic 863catgatcagc
tgggccaaga agaggga
2786416DNAArtificial SequenceSynthetic 864tccgagcctg ggtctc
1686514DNAArtificial
SequenceSynthetic 865tccgagcctg ggtc
1486630DNAArtificial SequenceSynthetic 866catgatcagc
tgggccaaga ctcggggcag
3086729DNAArtificial SequenceSynthetic 867catgatcagc tgggccaaga ctcggggca
2986828DNAArtificial
SequenceSynthetic 868catgatcagc tgggccaaga ctcggggc
2886927DNAArtificial SequenceSynthetic 869catgatcagc
tgggccaaga ctcgggg
2787017DNAArtificial SequenceSynthetic 870tcctgtactg agctgcc
1787115DNAArtificial
SequenceSynthetic 871tcctgtactg agctg
1587230DNAArtificial SequenceSynthetic 872catgatcagc
tgggccaaga aggtttcccg
3087329DNAArtificial SequenceSynthetic 873catgatcagc tgggccaaga aggtttccc
2987428DNAArtificial
SequenceSynthetic 874catgatcagc tgggccaaga aggtttcc
2887527DNAArtificial SequenceSynthetic 875catgatcagc
tgggccaaga aggtttc
2787616DNAArtificial SequenceSynthetic 876tgaaacatac acggga
1687714DNAArtificial
SequenceSynthetic 877tgaaacatac acgg
1487830DNAArtificial SequenceSynthetic 878catgatcagc
tgggccaaga ctggcacaca
3087929DNAArtificial SequenceSynthetic 879catgatcagc tgggccaaga ctggcacac
2988028DNAArtificial
SequenceSynthetic 880catgatcagc tgggccaaga ctggcaca
2888127DNAArtificial SequenceSynthetic 881catgatcagc
tgggccaaga ctggcac
2788215DNAArtificial SequenceSynthetic 882tgaaggtcta ctgtg
1588314DNAArtificial
SequenceSynthetic 883tgaaggtcta ctgt
1488430DNAArtificial SequenceSynthetic 884catgatcagc
tgggccaaga agcctatgga
3088529DNAArtificial SequenceSynthetic 885catgatcagc tgggccaaga agcctatgg
2988628DNAArtificial
SequenceSynthetic 886catgatcagc tgggccaaga agcctatg
2888727DNAArtificial SequenceSynthetic 887catgatcagc
tgggccaaga agcctat
2788818DNAArtificial SequenceSynthetic 888tgagaactga attccata
1888916DNAArtificial
SequenceSynthetic 889tgagaactga attcca
1689030DNAArtificial SequenceSynthetic 890catgatcagc
tgggccaaga tacacacttc
3089129DNAArtificial SequenceSynthetic 891catgatcagc tgggccaaga tacacactt
2989228DNAArtificial
SequenceSynthetic 892catgatcagc tgggccaaga tacacact
2889327DNAArtificial SequenceSynthetic 893catgatcagc
tgggccaaga tacacac
2789417DNAArtificial SequenceSynthetic 894tggaatgtaa agaagtg
1789515DNAArtificial
SequenceSynthetic 895tggaatgtaa agaag
1589630DNAArtificial SequenceSynthetic 896catgatcagc
tgggccaaga cactgtgggc
3089729DNAArtificial SequenceSynthetic 897catgatcagc tgggccaaga cactgtggg
2989828DNAArtificial
SequenceSynthetic 898catgatcagc tgggccaaga cactgtgg
2889927DNAArtificial SequenceSynthetic 899catgatcagc
tgggccaaga cactgtg
2790018DNAArtificial SequenceSynthetic 900tggtgcggag agggccca
1890115DNAArtificial
SequenceSynthetic 901tggtgcggag agggc
1590230DNAArtificial SequenceSynthetic 902catgatcagc
tgggccaaga actgtgggcc
3090329DNAArtificial SequenceSynthetic 903catgatcagc tgggccaaga actgtgggc
2990428DNAArtificial
SequenceSynthetic 904catgatcagc tgggccaaga actgtggg
2890527DNAArtificial SequenceSynthetic 905catgatcagc
tgggccaaga actgtgg
2790616DNAArtificial SequenceSynthetic 906tggtgcggaa agggcc
1690714DNAArtificial
SequenceSynthetic 907tggtgcggaa aggg
1490830DNAArtificial SequenceSynthetic 908catgatcagc
tgggccaaga gtagtgggcc
3090929DNAArtificial SequenceSynthetic 909catgatcagc tgggccaaga gtagtgggc
2991028DNAArtificial
SequenceSynthetic 910catgatcagc tgggccaaga gtagtggg
2891127DNAArtificial SequenceSynthetic 911catgatcagc
tgggccaaga gtagtgg
2791217DNAArtificial SequenceSynthetic 912tgtcactcgg ctcggcc
1791315DNAArtificial
SequenceSynthetic 913tgtcactcgg ctcgg
1591430DNAArtificial SequenceSynthetic 914catgatcagc
tgggccaaga agaggcaggc
3091529DNAArtificial SequenceSynthetic 915catgatcagc tgggccaaga agaggcagg
2991628DNAArtificial
SequenceSynthetic 916catgatcagc tgggccaaga agaggcag
2891727DNAArtificial SequenceSynthetic 917catgatcagc
tgggccaaga agaggca
2791818DNAArtificial SequenceSynthetic 918tgtctgcctg agtgcctg
1891916DNAArtificial
SequenceSynthetic 919tgtctgcctg agtgcc
1692030DNAArtificial SequenceSynthetic 920catgatcagc
tgggccaaga ttcagttatc
3092129DNAArtificial SequenceSynthetic 921catgatcagc tgggccaaga ttcagttat
2992228DNAArtificial
SequenceSynthetic 922catgatcagc tgggccaaga ttcagtta
2892327DNAArtificial SequenceSynthetic 923catgatcagc
tgggccaaga ttcagtt
2792418DNAArtificial SequenceSynthetic 924tgtgacagat tgataact
1892516DNAArtificial
SequenceSynthetic 925tgtgacagat tgataa
1692632DNAArtificial SequenceSynthetic 926catgatcagc
tgggccaaga cgtgacatga tg
3292729DNAArtificial SequenceSynthetic 927catgatcagc tgggccaaga cgtgacatg
2992828DNAArtificial
SequenceSynthetic 928catgatcagc tgggccaaga cgtgacat
2892927DNAArtificial SequenceSynthetic 929catgatcagc
tgggccaaga cgtgaca
2793016DNAArtificial SequenceSynthetic 930ctcggggatc atcatg
1693115DNAArtificial
SequenceSynthetic 931ctcggggatc atcat
1593230DNAArtificial SequenceSynthetic 932catgatcagc
tgggccaaga aaacatcact
3093329DNAArtificial SequenceSynthetic 933catgatcagc tgggccaaga aaacatcac
2993428DNAArtificial
SequenceSynthetic 934catgatcagc tgggccaaga aaacatca
2893527DNAArtificial SequenceSynthetic 935catgatcagc
tgggccaaga aaacatc
2793617DNAArtificial SequenceSynthetic 936ttaagacttg cagtgat
1793715DNAArtificial
SequenceSynthetic 937ttaagacttg cagtg
1593830DNAArtificial SequenceSynthetic 938catgatcagc
tgggccaaga gttagtggac
3093929DNAArtificial SequenceSynthetic 939catgatcagc tgggccaaga gttagtgga
2994028DNAArtificial
SequenceSynthetic 940catgatcagc tgggccaaga gttagtgg
2894127DNAArtificial SequenceSynthetic 941catgatcagc
tgggccaaga gttagtg
2794217DNAArtificial SequenceSynthetic 942tttgtgacct ggtccac
1794315DNAArtificial
SequenceSynthetic 943tttgtgacct ggtcc
1594430DNAArtificial SequenceSynthetic 944catgatcagc
tgggccaaga tccacatgga
3094529DNAArtificial SequenceSynthetic 945catgatcagc tgggccaaga tccacatgg
2994628DNAArtificial
SequenceSynthetic 946catgatcagc tgggccaaga tccacatg
2894727DNAArtificial SequenceSynthetic 947catgatcagc
tgggccaaga tccacat
2794816DNAArtificial SequenceSynthetic 948tgtaacagca actcca
1694917DNAArtificial
SequenceSynthetic 949tgtaacagca actccat
1795030DNAArtificial SequenceSynthetic 950catgatcagc
tgggccaaga ccacacactt
3095129DNAArtificial SequenceSynthetic 951catgatcagc tgggccaaga ccacacact
2995228DNAArtificial
SequenceSynthetic 952catgatcagc tgggccaaga ccacacac
2895327DNAArtificial SequenceSynthetic 953catgatcagc
tgggccaaga ccacaca
2795416DNAArtificial SequenceSynthetic 954tggaatgtaa ggaagt
1695518DNAArtificial
SequenceSynthetic 955tggaatgtaa ggaagtgt
1895629DNAArtificial SequenceSynthetic 956catgatcagc
tgggccaaga tacatactt
2995728DNAArtificial SequenceSynthetic 957catgatcagc tgggccaaga tacatact
2895827DNAArtificial
SequenceSynthetic 958catgatcagc tgggccaaga tacatac
2795917DNAArtificial SequenceSynthetic 959tggaatgtaa
agaagta
1796030DNAArtificial SequenceSynthetic 960catgatcagc tgggccaaga
tcatacagct 3096129DNAArtificial
SequenceSynthetic 961catgatcagc tgggccaaga tcatacagc
2996228DNAArtificial SequenceSynthetic 962catgatcagc
tgggccaaga tcatacag
2896327DNAArtificial SequenceSynthetic 963catgatcagc tgggccaaga tcataca
2796417DNAArtificial
SequenceSynthetic 964tctttggtta tctagct
1796520DNAArtificial SequenceSynthetic 965tctttggtta
tctagctgta
2096622RNAArtificial SequenceSynthetic 966aaacaaacau ggugcacuuc uu
2296723RNAArtificial
SequenceSynthetic 967aaagugcuuc cacuuugugu gcc
2396823RNAArtificial SequenceSynthetic 968aaagugcauc
cauuuuguuu guc
2396925RNAArtificial SequenceSynthetic 969aaagugcauc cuuuuagagu guuac
2597023RNAArtificial
SequenceSynthetic 970aaagugcauc cuuuuagagg uuu
2397122RNAArtificial SequenceSynthetic 971aaagugcauc
uuuuuagagg au
2297223RNAArtificial SequenceSynthetic 972caaagugccu cccuuuagag ugu
2397322RNAArtificial
SequenceSynthetic 973aaagugcuuc ccuuuggacu gu
2297421RNAArtificial SequenceSynthetic 974aaagugcuuc
cuuuuagagg g
2197523RNAArtificial SequenceSynthetic 975aaagugcuuc ucuuuggugg guu
2397621RNAArtificial
SequenceSynthetic 976aaagugcuuc cuuuuugagg g
2197722RNAArtificial SequenceSynthetic 977aagugcuucc
uuuuagaggg uu
2297822RNAArtificial SequenceSynthetic 978aacacaccca gcuaaccuuu uu
2297924RNAArtificial
SequenceSynthetic 979aacauucauu guugucggug gguu
2498024RNAArtificial SequenceSynthetic 980aacuggcccu
caaagucccg cuuu
2498124RNAArtificial SequenceSynthetic 981aauccuugga accuaggugu gagu
2498223RNAArtificial
SequenceSynthetic 982aauccuugga accuaggugu gaa
2398322RNAArtificial SequenceSynthetic 983augcaccugg
gcaaggauuc ug
2298422RNAArtificial SequenceSynthetic 984augcaccugg gcaaggguuc ag
2298522RNAArtificial
SequenceSynthetic 985aauccuuugu cccuggguga ga
2298622RNAArtificial SequenceSynthetic 986aauccuuugu
cccuggguga aa
2298721RNAArtificial SequenceSynthetic 987aaucguacag ggucauccac u
2198823RNAArtificial
SequenceSynthetic 988agugacauca cauauacggc agc
2398923RNAArtificial SequenceSynthetic 989aaugacacca
cauauauggc agc
2399023RNAArtificial SequenceSynthetic 990aaugacauca cauauauggc agc
2399123RNAArtificial
SequenceSynthetic 991aaugacacga ucacucccgu uga
2399223RNAArtificial SequenceSynthetic 992aauggcgcca
cuaggguugu gca
2399322RNAArtificial SequenceSynthetic 993agaggcuggc cgugaugaau uc
2299423RNAArtificial
SequenceSynthetic 994agucauacac ggcucuccuc ucu
2399521RNAArtificial SequenceSynthetic 995agaucgaccg
uguuauauuc g
2199624RNAArtificial SequenceSynthetic 996aggaagcccu ggaggggcug gagg
2499722RNAArtificial
SequenceSynthetic 997aggcagugua uuguuagcug gc
2299821RNAArtificial SequenceSynthetic 998aggcagugca
uugcuagcug g
2199920RNAArtificial SequenceSynthetic 999aggcagugcg accuggcucg
20100022RNAArtificial
SequenceSynthetic 1000agguuacccg agcaacuuug ca
22100123RNAArtificial SequenceSynthetic 1001gaauguugcu
cggugaaccc cuu
23100222RNAArtificial SequenceSynthetic 1002aguggggaac ccuuccauga gg
22100320RNAArtificial
SequenceSynthetic 1003auuccuagaa auuguucaua
20100420RNAArtificial SequenceSynthetic 1004auuccuagaa
auuguucaca
20100523RNAArtificial SequenceSynthetic 1005caaagugcuc auagugcagg uag
23100622RNAArtificial
SequenceSynthetic 1006caaccuggag gacuccaugc ug
22100721RNAArtificial SequenceSynthetic 1007cagcagcaca
cugugguuug u
21100824RNAArtificial SequenceSynthetic 1008cagugcaaug auauugucaa agca
24100924RNAArtificial
SequenceSynthetic 1009cagugcaaug guauugucaa agca
24101021RNAArtificial SequenceSynthetic 1010cagugcaauu
aaaaggggga a
21101122RNAArtificial SequenceSynthetic 1011caugccuuga guguaggacc gu
22101221RNAArtificial
SequenceSynthetic 1012cccagauaau ggcacucuca a
21101321RNAArtificial SequenceSynthetic 1013cccagauaau
agcacucuca a
21101422RNAArtificial SequenceSynthetic 1014ggagaaauua uccuuggugu gu
22101522RNAArtificial
SequenceSynthetic 1015gucaacacuu gcugguuucc uc
22101622RNAArtificial SequenceSynthetic 1016cgucaacacu
ugcugguuuu cu
22101722RNAArtificial SequenceSynthetic 1017uaaggugcau cuagugcagu ua
22101822RNAArtificial
SequenceSynthetic 1018uaaggugcau cuagugcugu ua
22101921RNAArtificial SequenceSynthetic 1019uagcagcggg
aacaguacug c
21102022RNAArtificial SequenceSynthetic 1020uaugugccuu uggacuacau cg
22102121RNAArtificial
SequenceSynthetic 1021uauugcacuc gucccggccu c
21102223RNAArtificial SequenceSynthetic 1022ucacuccucu
ccucccgucu ucu
23102323RNAArtificial SequenceSynthetic 1023ucacuccucc ccucccgucu ugu
23102422RNAArtificial
SequenceSynthetic 1024ucaggcucag uccccucccg au
22102524RNAArtificial SequenceSynthetic 1025ucccugagga
gcccuuugag ccug
24102621RNAArtificial SequenceSynthetic 1026uccgagccug ggucucccuc u
21102722RNAArtificial
SequenceSynthetic 1027uccuguacug agcugccccg ag
22102821RNAArtificial SequenceSynthetic 1028ugaaacauac
acgggaaacc u
21102921RNAArtificial SequenceSynthetic 1029ugaaggucua cugugugcca g
21103022RNAArtificial
SequenceSynthetic 1030ugagaacuga auuccauagg cu
22103121RNAArtificial SequenceSynthetic 1031uggaauguaa
agaagugugu a
21103223RNAArtificial SequenceSynthetic 1032uggugcggag agggcccaca gug
23103322RNAArtificial
SequenceSynthetic 1033uggugcggaa agggcccaca gu
22103423RNAArtificial SequenceSynthetic 1034ugucacucgg
cucggcccac uac
23103523RNAArtificial SequenceSynthetic 1035ugucugccug agugccugcc ucu
23103622RNAArtificial
SequenceSynthetic 1036ugugacagau ugauaacuga aa
22103721RNAArtificial SequenceSynthetic 1037cucggggauc
aucaugucac g
21103821RNAArtificial SequenceSynthetic 1038uuaagacuug cagugauguu u
21103922RNAArtificial
SequenceSynthetic 1039uuugugaccu gguccacuaa cc
22104022RNAArtificial SequenceSynthetic 1040uguaacagca
acuccaugug ga
22104122RNAArtificial SequenceSynthetic 1041uggaauguaa ggaagugugu gg
22104221RNAArtificial
SequenceSynthetic 1042uggaauguaa agaaguaugu a
21104323RNAArtificial SequenceSynthetic 1043ucuuugguua
ucuagcugua uga 23
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